Beta-lactamase inhibitors

ABSTRACT

This invention pertains generally to compounds of Formula (A), as further described herein, which act as beta-lactamase inhibitors, and salts, crystalline forms and formulations thereof. In certain aspects, the invention pertains to methods of using such compounds in combination with a beta-lactam antibiotic to treat infections caused by Gram-negative bacteria, including drug-resistant strains.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/401,022, filed 28 Sep. 2016, the content of which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to compounds that inhibit beta-lactamases,methods to make these compounds, and their use in combination withbeta-lactam antibiotics for treatment of bacterial infections.

BACKGROUND

Over the past several decades, the frequency of antimicrobial resistanceand its association with serious infectious diseases have increased atalarming rates. The increasing prevalence of resistance among nosocomialpathogens is particularly disconcerting. Of the over 2 millionnosocomial infections occurring each year in the United States, 50 to60% are caused by antimicrobial-resistant strains of bacteria. The highrate of resistance to commonly used antibacterial agents increases themorbidity, mortality, and costs associated with nosocomial infections.In the United States, nosocomial infections are thought to contribute toor cause more than 77,000 deaths per year and cost approximately $5 to$10 billion annually.

Among the most important antibiotics currently available are severalclasses of compounds that contain a beta-lactam ring, includingpenicillins, penems, carbapenems, cephalosporins, monobactams andsulfactams. These beta-lactam antibiotics inhibit cell wall biosynthesisby binding to proteins called penicillin-binding proteins (PBPs), whichare essential for synthesis of peptidoglycan, the major component of thecell wall of Gram-negative and Gram-positive bacteria. While beta-lactamantibiotics remain extremely important worldwide, their extensive usehas led to a large and growing problem: bacteria have developedresistance to beta-lactams, just as they have to most other availableantibiotics. Indeed, the World Health Organization (WHO) says antibioticresistance is a “serious, worldwide threat . . . ”

Several different mechanisms of resistance to beta-lactam antibioticshave been identified: some resistant strains possess efflux pumps toexcrete antibiotic, and others develop mutant PBPs that are lesssensitive to the antibiotic. An especially troubling form of resistanceis development of bacterial enzymes that react with these antibiotics,destroying the antibiotic by opening the beta-lactam ring. Theseantibiotic-degrading enzymes are called beta-lactamases, and areparticularly problematic because they can impart resistance to manydifferent beta-lactam antibiotics, and they can be transferred viaplasmids between different bacterial strains and species. AmongGram-negative bacteria, there are four classes of beta-lactamases, theserine beta-lactamases of the classes A, C and D, and the metallobeta-lactamases (class B).

Important causes of resistance to beta-lactam antibiotics includeextended-spectrum beta-lactamases (ESBLs), serine carbapenemases of theclass A, (e.g. KPC-2) and of class D (e.g. OXA-48) in Klebsiellapneumoniae, Escherichia coli, and Proteus mirabilis, as well ashigh-level resistance against third-generation cephalosporins mediatedby the class C beta-lactamase AmpC among Enterobacter species andCitrobacter freundii, and multidrug-resistance strains of Pseudomonas ,Acinetobacter, and Stenotrophomonas. The problem of antibacterialresistance is compounded by the existence of bacterial strainscontaining multiple beta-lactamases. For example, Klebsiella pneumoniaharboring NDM-1 metallo-beta-lactamase frequently carries additionalserine-beta-lactamases on the same plasmid that carries the NDM-1.

Since beta-lactam antibiotics are among the few classes that areeffective against Gram-negative bacteria, many efforts have been made tobolster their ability to control resistant bacterial strains, in orderto avoid losing these enormously valuable antibacterials. For example,some beta-lactams have been modified structurally to make them lesssusceptible to beta-lactamases, although this approach is complicated bythe fact that there are already many different beta-lactamases, and newones arise constantly. Another approach has been to inhibit thebeta-lactamase enzymes that degrade these antibiotics by using asmall-molecule beta-lactamase inhibitor (BLI) in combination with abeta-lactam antibiotic. These BLIs can be used in combination with anapproved beta-lactam antibiotic to treat patients infected with bacteriathat are resistant to the antibiotic alone due to beta-lactamaseactivity. Examples of approved BLIs include clavulanic acid, sulbactam,tazobactam, and avibactam. Others (relebactam, vaborbactam (RPX7009),zidebactam, and nacubactam) are reportedly in development.

In Gram-positive organisms, penicillin resistance mediated bypenicillinase-type beta-lactamases is an important mechanism ofresistance in Staphylococcus aureus (MSSA). Beta-lactamase-mediateresistance to penicillins is also found in anaerobic species, likebacteroides.

The three most commonly used serine beta-lactamase inhibitors,clavulanic acid, tazobactam and sulbactam, have potent activity onlyagainst some class A beta-lactamases, excluding serine carbapenemases.Avibactam is a member of the diazabicyclooctane (DBO) class ofbeta-lactamase inhibitors and has a broad coverage of class A (includingKPCs), class C and some inhibition of class D. Along with beta-lactamaseinhibition, avibactam also has antibacterial activity against someclinical strains through inhibiting penicillin binding protein 2 (PBP-2)(Asli et al, Antimicrobial Agents and Chemotherapy, 60, No 2, 752,2016). Antibacterial compounds with this mechanism of action, includingDBOs, select for resistance at very high frequencies in vitro (Doumithet al, J. Antimicrobial Chemotherapy 2016, 71, 2810-2814). Because ofthis, any potential clinical benefit of the intrinsic antibacterialactivity of some DBO beta-lactamase inhibitors is currently unclear. Theweak antibacterial activity of avibactam may not be clinically relevant,since the clinical dose of avibactam is fairly low, however, it maycomplicate in vitro susceptibility testing and/or promote resistance. Invitro susceptibility testing of avibactam/beta-lactam combinationsagainst clinical isolates is typically conducted using a high fixedconcentration of avibactam (4 μg/mL) that likely does not reflect theclinically achieved levels. The direct contribution of avibactam toantibacterial activity under these artificial in vitro testingconditions could affect the accuracy in predicting clinical efficacy ofavibactam/beta-lactamase combinations. A DBO beta-lactamase inhibitordevoid of significant antibacterial activity would not have this extraconfounding activity, and in vitro testing protocols would measure onlythe reversal of beta-lactamase mediated resistance in clinical isolates,enabling a more accurate prediction of clinical efficacy based on invitro susceptibility results.

In addition to BLIs currently available for use, other compounds withBLI activity are disclosed in WO2002/100860, US2003/0199541,US2004/0157826, WO2008/039420, and WO2009/091856, US2010092443,WO2010/126820, WO2013/122888, WO2013/038330, US2013/0225554,WO2013149121, WO2013149136, WO2014141132 and WO2014/033560.

The pharmacokinetic and physical properties of previously described BLIsmay not be ideal for use with every beta-lactam antibiotic. Moreover,known BLIs are reportedly losing effectiveness over time (K. Bush, Int.J. Antimicrob. Agents 46(5), 483-93 (November 2015)), as resistantbacterial strains develop and new beta-lactamase enzymes arisecontinually. Accordingly, there remains a need for new beta-lactamaseinhibitors to extend the usefulness of valuable beta-lactam antibiotics;indeed, novel BLIs may also combat resistance to known BLIs as well asresistance to known and future-developed beta-lactam antibiotics. Thepresent invention provides novel beta-lactamase inhibitors thatpotentiate the activity of various beta-lactam antibiotics, while theyexhibit little intrinsic (direct) antibiotic activity of their own.

SUMMARY

The invention includes novel BLI compounds, pharmaceutical combinationsand formulations including these compounds, and methods of using suchcompounds and compositions for treatment of patients with bacterialinfections. The BLIs are used in combination with a beta-lactamantibiotic, e.g. a penicillin derivatives, penem, carbapenem,cephalosporin (cephem), monobactam or sulfactam, and are primarilyuseful for treatment of Gram-negative bacterial infections, but also areuseful for the treatment of Gram-positive and anaerobic infections,where resistance is mediated through production of a beta-lactamase bythe bacterium. The invention includes compounds of Formula (A) andvariants thereof,

and the salts of these compounds, including compounds of Formula (I):

wherein compounds of Formula (I) may be in a salt or zwitterionic form,as further described herein.

The BLI compounds of the invention are used in combination with abeta-lactam antibiotic, examples of which are disclosed herein, to treatbacterial infections, especially Gram-negative bacterial infections. TheBLI and beta-lactam antibiotic can be administered together orseparately, and the BLI enhances the effectiveness of the beta-lactamantibiotic against at least on bacterial strains that exhibit resistanceto beta-lactam antibiotics, where the resistance is mediated by abeta-lactamase activity. The combinations of beta-lactam antibiotic andBLI of Formula (A) can be used to treat infections caused byGram-negative bacteria, including Enterobacteriaceae, such asSalmonella, E. coli, Klebsiella pneumoniae, Proteus, Enterobacter,Serratia, and Citrobacter, non-fermenting bacteria, includingPseudomonas aeruginosa, Acinetobacter, Burkholderia, Moraxella andStenotrophomonas, Gram-positive bacteria, such as beta-lactamaseproducing Staphylococcus aureus, as well as anaerobic bacteria, such asBacteroides fragilis or Bacteroides thetaiotaomicron.

In one aspect, the invention provides novel compounds of Formula (A) andFormula (I), including their salt or zwitterionic forms, which areeffective as inhibitors of one or more bacterial beta-lactamases. Thecompounds are useful to potentiate the antibacterial activity of abeta-lactam antibiotic. They may thus be used in combination with abeta-lactam antibiotic. The BLI and beta-lactam antibiotic may beadministered together or separately; in some embodiments, a BLI ofFormula (A) or Formula (I) and a beta-lactam antibiotic are combined ina pharmaceutical composition that typically also comprises at least onepharmaceutically acceptable carrier.

In one aspect, the invention provides methods to make compounds ofFormula (A) or Formula (I) and novel precursors useful to make compoundsof Formula (A) or Formula (I) as described herein. In particular, theinvention provides a process to convert a compound of Formula (V) into acompound of Formula (IV); and a method to convert a compound of Formula(III) into compounds of Formula (I).

In another aspect, the invention provides pharmaceutical compositionscomprising a compound of Formula (A) and Formula (I) admixed with atleast one pharmaceutically acceptable carrier or excipient. In someembodiments, the composition comprises two or more such carriers orexcipients. Optionally, the pharmaceutical composition further includesa beta-lactam antibiotic, although the BLI compound can be formulatedand administered separately from the beta-lactam antibiotic.

In another aspect, the invention provides a method for treating asubject having a bacterial infection, which comprises administering tothe subject in need thereof an antibacterially effective amount of abeta-lactam antibiotic and an effective amount of a BLI of Formula (A)or Formula (I), including salt and zwitterionic forms, optionally incombination with a pharmaceutically acceptable carrier. In certainembodiments, the subject is a mammal and in some embodiments, thesubject is a human. This aspect provides a compound of Formula (A) orFormula (I), including pharmaceutically acceptable salt or zwitterionicforms, for use to treat a bacterial infection, where the compound isused in combination with a beta-lactam antibiotic. It also includes useof a compound of Formula (A) or Formula (I), or a salt or zwitterionicform thereof, in the manufacture of a medicament. Preferably, themedicament is one for use to treat a Gram-negative bacterial infection,especially one having a beta-lactamase activity sufficient to impartsome level of resistance to the beta-lactam antibiotic, where themedicament is adapted for use in combination with a beta-lactamantibiotic such as those described herein. The beta-lactam antibioticand BLI compound of Formula (A) or Formula (I) may be administeredsimultaneously or separately and in any order, provided the BLI ispresent in vivo concurrently with the beta-lactam antibiotic in order topotentiate the effectiveness of the beta-lactam antibiotic.

The Gram-negative bacteria may be of a genus selected from Citrobacter,Enterobacter, Escherichia, Klebsiella, Morganella, Proteus, Salmonella,Serratia, Pseudomonas, Acinetobacter, Bacteroides, Burkholderia,Campylobacter, Neisseria, and Stenotrophomonas. In particular, abacterial infection caused by a species of Citrobacter, Enterobacter,Escherichia, Klebsiella, Morganella, Proteus, Salmonella, Serratia,Pseudomonas, or Acinetobacter is treatable by the methods disclosedherein. Particular bacterial species for such treatment includeCitrobacter freundii, Citrobacter koseri, Enterobacter cloacae,Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae,Klebsiella oxytoca, Morganella morganii, Proteus mirabilis, Salmonellaspecies, Serratia marcescens, Pseudomonas aeruginosa, and Acinetobacterbaumannii, as well as Bacteroides fragilis, Burkholderia cepacia,Campylobacter jejuni, Neisseria gonorrhoeae, and Stenotrophomonasmaltophilia. The Gram-positive bacteria may be, for example,Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus orStreptococcus pneumoniae.

In another aspect, the invention provides a method of inhibitingbacterial growth or modulating the virulence of a bacterial infection,wherein the method comprises administering to a patient in need of suchinhibition a compound of Formula (A) or Formula (I) and a beta-lactamantibiotic. Suitable beta-lactam antibiotics for use in these methodsare described herein.

Pharmaceutical compositions according to the present invention areprovided which include any of the compounds described herein and apharmaceutically acceptable carrier. In some embodiments the compositionincludes an additional therapeutic agent such as a beta-lactamantibiotic.

Other aspects of the invention are discussed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. SEM of Crystalline Compound of Formula (VII).

FIG. 2. XRPD of Crystalline Compound of Formula (VII).

FIG. 3. Thermogravimetric Analysis and Differential Scanning CalorimetryAnalysis of Crystalline Compound of Formula (VII).

DETAILED DESCRIPTION

For purposes of interpreting this specification, the followingdefinitions apply unless specified otherwise or clearly contradicted bycontext. Whenever appropriate, terms used in the singular will alsoinclude the plural and vice versa.

Definitions

Terms used in the specification have the following meanings:

As used herein, the term “subject” refers to an animal. In certainaspects, the animal is a mammal. A subject also refers to for example,primates (e.g., humans), cows, sheep, goats, horses, dogs, cats,rabbits, rats, mice, fish, birds and the like. In certain embodiments,the subject is a human.

As used herein, the term “inhibition” or “inhibiting” refers to thereduction or suppression of a given condition, symptom, or disorder, ordisease, or a significant decrease in the baseline activity of abiological activity or process, or decrease in the viability, number orgrowth rate of a bacterial population.

As used herein, the term “treating” or “treatment” of any disease ordisorder refers in one embodiment, to ameliorating the disease ordisorder (i.e., slowing or arresting or reducing the development of thedisease or at least one of the clinical symptoms thereof). In anotherembodiment “treating” or “treatment” refers to alleviating orameliorating at least one physical parameter including those which maynot be discernible by the patient. In yet another embodiment, “treating”or “treatment” refers to modulating the disease or disorder, eitherphysically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In yet another embodiment, “treating” or “treatment” refers topreventing or delaying the onset or development or progression of thedisease or disorder.

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

The term “antibacterial agent” refers to agents synthesized or modifiedin the laboratory that have either bactericidal or bacteriostaticactivity. An “active” agent in this context will inhibit the growth ofP. aeruginosa and/or other Gram-negative bacteria. The term “inhibitingthe growth” indicates that the rate of increase in the numbers of apopulation of a particular bacterium is reduced. Thus, the term includessituations in which the bacterial population increases but at a reducedrate, as well as situations where the growth of the population isstopped, as well as situations where the numbers of the bacteria in thepopulation are reduced or the population even eliminated.

The term “beta-lactam antibiotic” refers to an antibacterial agent thatcontains a 4-membered lactam ring, also referred to as a beta-lactam,that possesses antibacterial activity. Classes of beta-lactamantibiotics include penicillins, cephalosporins, monobactams,carbapenems, oxapenems, cephems, carbacephems, oxacephems, penems,penams, sulbactams and clavams. Particular beta-lactam antibioticssuitable for use in the methods and compositions of the invention aredescribed herein, and include aztreonam, piperacillin, ceftazidime,meropenem, and beta-lactam 5.

The term “beta-lactamase” as used herein refers to an enzymatic activitypossessed or exhibited by a bacterium, which catalyzes the degradationor inactivation of a beta-lactam antibiotic. Typically, it catalyzeshydrolysis of the beta-lactam ring of a monocyclic or bicyclicbeta-lactam antibiotic, and is expressed in a Gram-negative orGram-positive bacterium that can cause infection in mammalian subjects,especially humans. Beta-lactamases of interest include Class A(including extended spectrum beta-lactamases and serine carbapenemases),as well as Class C and D beta-lactamases.

The term “beta-lactamase inhibitor” or “BLI” as used herein refers to acompound that inhibits at least one bacterial beta-lactamase. This meansit inhibits at least one member of the classes of serinebeta-lactamases, e.g. a Class A, C or D beta-lactamase. By reducingbeta-lactamase activity, these compounds enhance the activity of abeta-lactam antibiotic used in combination with the BLI; this effect isreferred to herein as potentiation, since the BLI does not havesignificant antibacterial activity of its own but boosts or potentiatesthe antibacterial activity of the antibiotic in bacteria that possessbeta-lactamase activity. Potentiation results from the fact that the BLIallows the beta-lactam antibiotic to persist longer in vivo within thebacterial periplasmic compartment or in the vicinity of the bacterialpathogens, making the antibiotic more effective, or making it effectiveat a lower dosage than would be required in the absence of the BLI ofFormula (A). Preferably, a BLI is effective at a 50% inhibitoryconcentration below about 100 μg/mL (micrograms/mL), or below about 50μg/mL, or below about 25 μg/mL.

Suitable beta-lactam antibiotics for use in combination with the BLIs ofthe invention include, for example, aztreonam, imipenem, ertapenem,meropenem, doripenem, biapenem, piperacillin, ceftriaxone, cefoperazone,cefotaxime, ceftazidime, ceftolozane, cefepime, panipenem, ticarcillin,ampicillin, amoxicillin, carbenicillin, azlocillin, mezlocillin,ticarcillin, cefoperazone, beta-lactam 5 (shown herein), and the like.

“Optionally substituted” means the group referred to can be substitutedat one or more positions by any one or any combination of the radicalslisted thereafter. Such substitution involves the replacement of ahydrogen atom of the unsubstituted group with another moiety; thus thenumber of substituents that can be added to any unsubstituted group isequal to the number of hydrogen atoms on the unsubstituted group. If nototherwise specified, ‘optionally substituted’ means that up to threenon-hydrogen substituent groups can be present.

“Halo” or “halogen”, as used herein, may be fluorine, chlorine, bromineor iodine.

“C₁-C₆ alkyl”, or “C₁₋₆ alkyl” as used herein, denotes straight chain orbranched alkyl having 1-6 carbon atoms. If a different number of carbonatoms is specified, such as C₈ or C₃, then the definition is to beinterpreted accordingly, such as “C₁-C₄ alkyl” will include methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.

“C₁-C₆ alkoxy”, or “C₁₋₆ alkoxy” as used herein, denotes straight chainor branched alkoxy having 1-6 carbon atoms. If a different number ofcarbon atoms is specified, such as C₈ or C₃, then the definition is tobe interpreted accordingly, e.g., “C₁-C₄ alkoxy” will represent methoxy,ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy andtert-butoxy.

“C₁-C₄-Haloalkyl” or “C₁₋₄ haloalkyl” as used herein, denotes straightchain or branched alkyl having 1-4 carbon atoms, wherein at least onehydrogen has been replaced by a halogen. If a different number of carbonatoms is specified, such as C₆ or C₃, then the definition is to beinterpreted accordingly, thus “C₁-C₄-Haloalkyl” will represent methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl thathave at least one hydrogen substituted with halogen, such as where thehalogen is fluorine: CF₃CF₂—, (CF₃)₂CH—, CH₃—CF₂—, CF₃CF₂—, CF₃, CF₂H—,CF₃CF₂CHCF₃ or CF₃CF₂CF₂CF₂—.

“C₃-C₈-Cycloalkyl” or “C₃₋₈ cycloalkyl” as used herein refers to asaturated monocyclic hydrocarbon ring of 3 to 8 carbon atoms. Examplesof such groups include cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl. If a different number of carbon atoms is specified, such asC₃-C₆, then the definition is to be interpreted accordingly.

“4- to 8-Membered heterocyclyl”, “5- to 6-membered heterocyclyl”, “3- to10-membered heterocyclyl”, “3- to 14-membered heterocyclyl”, “4- to14-membered heterocyclyl” and “5- to 14-membered heterocyclyl”, refer,respectively, to 4- to 8-membered, 5- to 6-membered, 3- to 10-membered,3- to 14-membered, 4- to 14-membered and 5- to 14-membered heterocyclicrings containing 1 to 7, 1 to 5 or 1 to 4 heteroatoms selected from thegroup consisting of nitrogen, oxygen and sulphur, which may besaturated, or partially saturated. “Heterocyclic” may be usedinterchangeably with “heterocyclyl”. The heterocyclic group can beattached at a heteroatom or a carbon atom. The term “heterocyclyl”includes single ring groups, fused ring groups and bridged groups.Examples of such heterocyclyl include, but are not limited topyrrolidine, piperidine, piperazine, oxazolidine, pyrrolidinone,morpholine, tetrahydrofuran, tetrahydrothiophene, tetrahydrothiopyran,tetrahydropyran, 1,4-dioxane, 1,4-oxathiane, 8-aza-bicyclo[3.2.1]octane,3,8-diazabicyclo[3.2.1]octane, 3-Oxa-8-aza-bicyclo[3.2.1]octane,8-Oxa-3-aza-bicyclo[3.2.1]octane, 2-Oxa-5-aza-bicyclo[2.2.1]heptane,2,5-Diaza-bicyclo[2.2.1]heptane, azetidine, ethylenedioxo, oxetane andthiazolidine. Preferably, a heterocyclic or heterocyclyl group is asaturated or partially saturated monocyclic group unless otherwisespecified, and contains 5-7 ring atoms with up to two heteroatomsselected from N, O and S as ring members. In some embodiments, aheterocyclic group further includes bicyclic ring systems containing 1or 2 heteroatoms such as N, O or S as ring members and comprising twofused 3-, 4-, 5-, or 6-membered rings, such as3-azabicyclo[3.1.0]hexane, 8-aza-bicyclo[3.2.1]octane,3,8-diazabicyclo[3.2.1]octane, 3-Oxa-8-aza-bicyclo[3.2.1]octane,8-Oxa-3-aza-bicyclo[3.2.1]octane, 2-Oxa-5-aza-bicyclo[2.2.1]heptane,2,5-Diaza-bicyclo[2.2.1]heptane.

“Heteroaryl” is a completely unsaturated (aromatic) ring. The term“heteroaryl” refers to a 5-14 membered monocyclic- or bicyclic- ortricyclic-aromatic ring system, having 1 to 8 heteroatoms selected fromN, O and S. Typically, the heteroaryl is a 5-10 membered ring system(e.g., 5-6 membered monocycle or an 8-10 membered bicycle) or a 5-6membered ring system. Unless otherwise specified, a heteroaryl ispreferably an isolated 5-6 membered ring containing up to 4 heteroatomsselected from N, O and S as ring members. Typical heteroaryl groupsinclude furan, isothiazole, thiadiazole, oxadiazole, indazole, indole,quinoline, 2- or 3-thienyl; 2- or 3-furyl; 2- or 3-pyrrolyl; 1-, 2-, 4-,or 5-imidazolyl; 1, 3-, 4-, or 5- pyrazolyl; 2-, 4-, or 5-thiazolyl, 3-,4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl,3- or 5-(1,2,4-triazolyl), 4- or 5-(1,2, 3-triazolyl), tetrazolyl,triazine, pyrimidine, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-,or 5-pyrazinyl, 2-pyrazinyl, and 2-, 4-, or 5-pyrimidinyl.

The term “hydroxy” or “hydroxyl” refers to the group —OH, or when usedas part of a group name such as hydroxyalkyl, it refers to the namedgroup substituted with an —OH.

A “zwitterion” is a molecule that has both positively-charged andnegatively-charged groups but has no overall charge i.e. the + and −charges are balanced within the molecule. To convert an anionic moleculeinto a neutral molecule then anions would typically be replaced byneutral groups, but to convert an anionic molecule into a zwitterionthen a neutral group is replaced by a cationic group.

Compounds of formula (A) exist in free form, as a salt or as zwitterion.In this specification, unless otherwise indicated, language such as“compounds of formula (A)” is to be understood as embracing thecompounds in any form, for example free base or acid addition orexchange salt form. Salts which may be unsuitable for pharmaceuticaluses but which can be employed, for example, for the isolation orpurification of free compounds of formula (A), such as picrates orperchlorates, are also included. For therapeutic use, onlypharmaceutically acceptable salts, zwitterions or free compounds areemployed (where applicable in the form of pharmaceutical preparations),and are therefore preferred. Salts are preferably physiologicallyacceptable salts, formed, as applicable, by the addition of an acid orbase or by ion exchange.

Compounds of formula (A) may exist in the form of various zwitterions.For example, the compounds of formula (A) may show protonatedamino-groups and deprotonated sulfate-groups. In this specification, thedrawing of the compound in the free form includes other possiblezwitterions as well. The zwitterions of the compounds of formula (A) arealso embraced by the invention.

The compounds of formula (A) may exist in optically active form or inform of mixtures of optical isomers, e.g. in form of racemic mixtures ordiastereomeric mixtures. In particular, asymmetrical carbon atom(s) maybe present in the compounds of formula (A) and their salts. All opticalisomers and their mixtures, including the racemic mixtures, are embracedby the invention. Various embodiments of the invention are describedherein. It will be recognized that features specified in each embodimentmay be combined with other specified features to provide furtherembodiments. The following numbered embodiments are representative ofadditional aspects of the invention.

In one embodiment, the invention provides compounds of Formula (A):

wherein p is 1 or 2;

R¹ and R² are independently selected from H and C₁-C₄ alkyl optionallysubstituted with up to three groups selected from halo, CN, —OR, oxo,and —NRR′;

Z is NR³ or N—OR³;

R³ is independently selected at each occurrence from H, Cy, and C₁-C₄alkyl optionally substituted with up to three groups selected from Cy,halo, CN, —OR, and —NRR′;

Cy is a C₃-C₆ cycloalkyl ring or 4-6 membered heterocyclic ringcontaining one or two heteroatoms selected from N, O and S as ringmembers, and Cy is optionally substituted with up to three groupsselected from oxo, halo, C₁-C₂ alkyl, CN, —OR, and —NRR′; and

R and R′ are independently selected from H and C₁-C₄ alkyl optionallysubstituted with one or two groups selected from halo, —OH, —CN,—O—(C₁-C₄ alkyl), oxo, —NH₂, —NH(C₁-C₄ alkyl), and —N(C₁-C₄ alkyl)₂, orR and R′ taken together with the nitrogen atom to which both areattached can form a ring selected from piperidine, morpholine,pyrrolidine, and azetidine, wherein the ring is optionally substitutedwith one or two groups selected from halo, CN, —OR, and —NRR′; alkyl,—OH, —CN, —O—(C₁-C₄ alkyl), oxo, —NH₂, —NH(C₁-C₄ alkyl), and —N(C₁-C₄alkyl)₂;

or a salt or zwitterionic form thereof.

Particular embodiments of these compounds include compounds thefollowing formulas:

or

or a salt or zwitterionic form thereof.

An embodiment of special interest is compound of Formula (I):

wherein:

R¹ and R² are independently selected from H and C₁-C₄ alkyl optionallysubstituted with up to three groups selected from halo, CN, —OR, oxo,and —NRR′;

Z is NR³ or N—OR³;

R³ is independently selected at each occurrence from H, Cy, and C₁-C₄alkyl optionally substituted with up to three groups selected from Cy,halo, CN, —OR, and —NRR′;

Cy is a C₃-C₆ cycloalkyl ring, or 4-6 membered heterocyclic ringcontaining one or two heteroatoms selected from N, O and S as ringmembers, and Cy is optionally substituted with up to three groupsselected from oxo, halo, C₁-C₂ alkyl, CN, —OR, and —NRR′; and

R and R′ are independently selected from H and C₁-C₄ alkyl optionallysubstituted with one or two groups selected from halo, —OH, —CN,—O—(C₁-C₄ alkyl), oxo, —NH₂, —NH(C₁-C₄ alkyl), and —N(C₁-C₄ alkyl)₂, orR and R′ taken together with the nitrogen atom to which both areattached can form a ring selected from piperidine, morpholine,pyrrolidine, and azetidine, wherein the ring is optionally substitutedwith one or two groups selected from halo, C₁-C₂ alkyl, —OH, —CN,—O—(C₁-C₄ alkyl), oxo, —NH₂, —NH(C₁-C₄ alkyl), and —N(C₁-C₄ alkyl)₂;

Y is a cationic group;

n is 0 or 1; and

when n is 0 the compound of Formula I is in a zwitterionic form.

Each of the compounds of the Examples herein is a specific embodiment ofthe invention.

The compound of any of the preceding embodiments, wherein Z is NR³

and R³ is H or C₁-C₄ alkyl optionally substituted with —OR or —NRR′, ora salt or zwitterionic form thereof.

The compound of embodiment 4, wherein R³ is C₁-C₂ alkyl optionallysubstituted with —OR or —NRR′, or a salt or zwitterionic form thereof.

The compound of embodiment 4, wherein R³ is H, or a salt or zwitterionicform thereof.

The compound of any one of embodiments 1-6, wherein R¹ and R² are bothH, or a salt or zwitterionic form thereof.

The compound of embodiment 1, which has this structure:

wherein X is —OR or —NRR′;

Y is a cationic group;

n is 0 or 1; and

when n is 0 the compound of Formula II is in a zwitterionic form.

The compound of embodiment 1, which is selected from:

as a salt or zwitterionic form thereof.

The compound of any of the preceding embodiments, wherein n is 1 and Yis selected from sodium, potassium, ammonium, calcium, magnesium, iron,silver, zinc, and copper.

The compound of any of the preceding embodiments, wherein Y is sodium.

The compound of any of the preceding embodiments, which is apharmaceutically acceptable salt or zwitterion.

An embodiment of special interest is a compound of Formula (VI):

wherein:

R¹ and R² are independently selected from H and C₁-C₄ alkyl optionallysubstituted with up to three groups selected from halo, CN, —OR, oxo,and —NRR′;

Z is NR³ or N—OR³;

R³ is independently selected at each occurrence from H, Cy, and C₁-C₄alkyl optionally substituted with up to three groups selected from Cy,halo, CN, —OR, and —NRR′;

Cy is a C₃-C₆ cycloalkyl ring or 4-6 membered heterocyclic ringcontaining one or two heteroatoms selected from N, O and S as ringmembers, and Cy is optionally substituted with up to three groupsselected from oxo, halo, C₁-C₂ alkyl, CN, —OR, and —NRR′; and

R and R′ are independently selected from H and C₁-C₄ alkyl optionallysubstituted with one or two groups selected from halo, C₁-C₂ alkyl, —OH,—CN, —O—(C₁-C₄ alkyl), oxo, —NH₂, —NH(C₁-C₄ alkyl), and —N(C₁-C₄alkyl)₂, or R and R′ taken together with the nitrogen atom to which bothare attached can form a ring selected from piperidine, morpholine,pyrrolidine, and azetidine, wherein the ring is optionally substitutedwith one or two groups selected from halo, C₁-C₂ alkyl, —OH, —CN,—O—(C₁-C₄ alkyl), oxo, —NH₂, —NH(C₁-C₄ alkyl), and —N(C₁-C₄ alkyl)₂;

A is H or —CH₂-Ph, where Ph represents phenyl optionally substitutedwith one or two groups selected from halo, C₁-C₄ alkyl, C₁-C₄ alkoxy; ora salt or zwitterion thereof.

An embodiment of special interest is a compound of the formula (VII):

The compound of embodiment 12 in crystalline form.

The compound of embodiment 13, which exhibits an endotherm ondifferential scanning calorimetry between 283° C. and 350° C.

The compound of embodiment 13, characterized by XRPD peaks atdiffraction angles (2Theta) of 8.3 and 16.6 degrees.

The compound of embodiment 15, further characterized by one or moreadditional XRPD peaks at diffraction angles (2Theta) of 25.1 or 31.3degrees.

The compound of embodiment 16, further characterized by one or moreadditional XRPD peaks at diffraction angles (2Theta) of 27.4 or 28.7degrees.

The compound of embodiment 17, further characterized by additional XRPDpeaks at diffraction angles (2Theta) of 19.5 degrees or 21.7 degrees.

A process to make a compound of Formula (I),

according to embodiment 3, as a salt or zwitterionic form thereof;

wherein the process comprises contacting a compound of Formula (III)

wherein Z, R¹ and R² and R³ are as defined in embodiment 3,

with a sulfonylating agent in the presence of a base.

The process of embodiment 19, wherein Z is NR³, and R³ is H or C₁-C₂alkyl optionally substituted with —OR or —NRR′,

or a pharmaceutically acceptable salt thereof.

The process of embodiment 19 or 20, wherein the compound of Formula (I)is of the formula

or a salt or zwitterionic form thereof.

The process of any one of embodiments 19 to 21, wherein R³ is H.

The compounds of Formula (III) are useful for synthesizing compounds ofFormula (I) as described in Embodiment 3 and other embodiments above.

Specific compounds of Formula (A) and Formula (I) include:

or a salt or zwitterion thereof.

In a further aspect, the invention provides:

A pharmaceutical combination comprising (a) a first therapeutic agentwhich is a compound of the invention, e.g. a compound of formula (A) orany subformula thereof described herein, and (b) a second therapeuticagent as described above. The second therapeutic agent is typically abeta-lactam antibiotic.

A method as defined above comprising co-administration, e.g.concomitantly or in sequence, of a therapeutically effective amount of acompound of the invention, e.g. a compound of formula (A) or anysubformulae thereof that is described herein, and a second therapeuticagent as described above.

The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time. Fixedcombinations are also within the scope of the present invention. Theadministration of a pharmaceutical combination of the invention resultsin a beneficial effect, e.g. a synergistic therapeutic effect, comparedto a monotherapy applying only one of its pharmaceutically activeingredients.

Each component of a combination according to this invention may beadministered separately, together, or in any combination thereof.

The compound of the invention and any additional agent may be formulatedin separate dosage forms. Alternatively, to decrease the number ofdosage forms administered to a patient, the compound of the inventionand any additional agent may be formulated together in any combination.For example, the compound of the invention inhibitor may be formulatedin one dosage form and the additional agent may be formulated togetherin another dosage form. Any separate dosage forms may be administered atthe same time or different times.

Alternatively, a composition of this invention comprises an additionalagent as described herein. Each component may be present in individualcompositions, combination compositions, or in a single composition.

The compounds of the invention may be synthesized by the generalsynthetic routes below, specific examples of which are described in moredetail in the Examples.

Compounds of the present invention and intermediates can also beconverted into each other according to methods generally known to thoseskilled in the art.

Within the scope of this text, only a readily removable group that isnot a constituent of the particular desired end product of the compoundsof the present invention is designated a “protecting group”, unless thecontext indicates otherwise. The protection of functional groups by suchprotecting groups, the protecting groups themselves, and their cleavagereactions are described for example in standard reference works, such asJ. F. W. McOmie, “Protective Groups in Organic Chemistry”, Plenum Press,London and New York 1973, in T. W. Greene and P. G. M. Wuts, “ProtectiveGroups in Organic Synthesis”, Third edition, Wiley, New York 1999, in“The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), AcademicPress, London and New York 1981, in “Methoden der organischen Chemie”(Methods of Organic Chemistry), Houben Weyl, 4th edition, Volume 15/I,Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jeschkeit,“Aminosauren, Peptide, Proteine” (Amino acids, Peptides, Proteins),Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in JochenLehmann, “Chemie der Kohlenhydrate: Monosaccharide und Derivate”(Chemistry of Carbohydrates: Monosaccharides and Derivatives), GeorgThieme Verlag, Stuttgart 1974. A characteristic of protecting groups isthat they can be removed readily (i.e. without the occurrence ofundesired secondary reactions) for example by solvolysis, reduction,photolysis or alternatively under physiological conditions (e.g. byenzymatic cleavage).

Salts of compounds of the present invention having at least onesalt-forming group may be prepared in a manner known to those skilled inthe art. For example, salts of compounds of the present invention havingacid groups may be formed, for example, by treating the compounds, or asalt of the compounds like the tetrabutylammonium salt, with metalcompounds, such as alkali metal salts of suitable organic carboxylicacids, e.g. the sodium salt of 2-ethylhexanoic acid in appropriatesuitable solvent, such as an isobutanol/water mixture, which mayfacilitate the undesired ion pair (e.g. the tetrabutyl ammonium2-ethylhexanoate if the tetrabutyl ammonium salt is used) toprecipitate. Preferably, a salt of a compound of the invention, such asthe ammonium salt, may be subjected to an ion exchange resin in itsalkali metal or alkaline earth metal form to promote a counterionexchange. Acid addition or exchange salts of compounds of the presentinvention are obtained in customary manner, e.g. by treating thecompounds with an acid or a suitable anion exchange reagent. Zwitterionsor internal salts of compounds of the present invention containing acidand basic salt-forming groups, e.g. a free sulfate group and a freeamino group, may be formed, e.g. by the neutralization of salts, such asacid addition salts, to the isoelectric point, e.g. with weak bases, orby treatment with ion exchangers.

Salts can be converted into the free compounds in accordance withmethods known to those skilled in the art. Hydrochloride salts can beconverted, for example, by treatment with a suitable basic agent.Mixtures of isomers obtainable according to the invention can generallybe separated in a manner known to those skilled in the art into theindividual isomers; diastereoisomers can be separated, for example, bypartitioning between polyphasic solvent mixtures, recrystallizationand/or chromatographic separation, for example over silica gel or bye.g. medium pressure liquid chromatography over a reversed phase column,and racemates can be separated, for example, by the formation of saltswith optically pure salt-forming reagents and separation of the mixtureof diastereoisomers so obtainable, for example by means of fractionalcrystallization, or by chromatography over optically active columnmaterials.

Intermediates and final products can be worked up and/or purifiedaccording to standard methods, e.g. using chromatographic methods,distribution methods, (re-) crystallization, and the like.

The following applies in general to all processes mentioned hereinbefore and hereinafter.

All process steps for making compounds of the invention can be carriedout under reaction conditions that are known to those skilled in theart, including those mentioned specifically, in the absence or,customarily, in the presence of solvents or diluents, including, forexample, solvents or diluents that are inert towards the reagents usedand dissolve them, in the absence or presence of catalysts, condensationor neutralizing agents, for example ion exchangers, such as cationexchangers, e.g. in the H+ form, depending on the nature of the reactionand/or of the reactants at reduced, normal or elevated temperature, forexample in a temperature range of from about −100° C. to about 190° C.,including, for example, from approximately −80° C. to approximately 150°C., for example at from −80 to −60° C., at room temperature, at from −20to 40° C. or at reflux temperature, under atmospheric pressure or in aclosed vessel, where appropriate under pressure, and/or in an inertatmosphere, for example under an argon or nitrogen atmosphere.

At all stages of the reactions, mixtures of isomers that are formed canbe separated into the individual isomers, for example diastereoisomersor enantiomers, or into any desired mixtures of isomers, for exampleracemates or mixtures of diastereoisomers

The solvents from which those solvents that are suitable for anyparticular reaction may be selected include those mentioned specificallyor, for example, water, esters, such as lower alkyl-lower alkanoates,for example ethyl acetate, ethers, such as aliphatic ethers, for examplediethyl ether, or cyclic ethers, for example tetrahydrofuran or dioxane,liquid aromatic hydrocarbons, such as benzene or toluene, alcohols, suchas methanol, ethanol or 1- or 2-propanol, nitriles, such asacetonitrile, halogenated hydrocarbons, such as methylene chloride orchloroform, acid amides, such as dimethylformamide or dimethylacetamide, bases, such as heterocyclic nitrogen bases, for examplepyridine or N-methylpyrrolidin-2-one, carboxylic acid anhydrides, suchas lower alkanoic acid anhydrides, for example acetic anhydride, cyclic,linear or branched hydrocarbons, such as cyclohexane, hexane orisopentane, methylcyclohexane, or mixtures of those solvents, forexample aqueous solutions, unless otherwise indicated in the descriptionof the processes. Such solvent mixtures may also be used in working up,for example by chromatography or partitioning.

The compounds of the present invention, including their salts, may alsobe obtained in the form of hydrates, or their crystals may, for example,include the solvent used for crystallization. Different crystallineforms may be present.

All starting materials, building blocks, reagents, acids, bases,dehydrating agents, solvents and catalysts utilized to synthesize thecompounds of the present invention are either commercially available orcan be produced by organic synthesis methods known to one of ordinaryskill in the art.

The term “optical isomer” or “a stereoisomer” refers to any of thevarious stereoisomeric configurations which may exist for a givencompound of the present invention and includes geometric isomers. It isunderstood that a substituent may be attached at a chiral center of acarbon atom. The term “chiral” refers to molecules which have theproperty of non-superimposability on their mirror image partner, whilethe term “achiral” refers to molecules which are superimposable on theirmirror image partner. Therefore, the invention includes enantiomers,diastereomers or racemates of the compound. “Enantiomers” are a pair ofstereoisomers that are non-superimposable mirror images of each other. A1:1 mixture of a pair of enantiomers is a “racemic” mixture. The term isused to designate a racemic mixture where appropriate.“Diastereoisomers” are stereoisomers that have at least two asymmetricatoms, but which are not mirror-images of each other. The absolutestereochemistry is specified according to the Cahn-Ingold-Prelog R-Ssystem. When a compound is a pure enantiomer the stereochemistry at eachchiral carbon may be specified by either R or S. Resolved compoundswhose absolute configuration is unknown can be designated (+) or (−)depending on the direction (dextro- or levorotatory) which they rotateplane polarized light at the wavelength of the sodium D line. Certaincompounds described herein contain one or more asymmetric centers oraxes and may thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-.

Depending on the choice of the starting materials and procedures, thecompounds can be present in the form of one of the possible isomers oras mixtures thereof, for example as pure optical isomers, or as isomermixtures, such as racemates and diastereoisomer mixtures, depending onthe number of asymmetric carbon atoms. The present invention is meant toinclude all such possible stereoisomers, including racemic mixtures,diasteriomeric mixtures and optically pure forms. Optically active (R)-and (S)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques. If the compoundcontains a double bond, the substituent may be E or Z configuration. Ifthe compound contains a disubstituted cycloalkyl, the cycloalkylsubstituent may have a cis- or trans-configuration. All tautomeric formsare also intended to be included.

Any resulting mixtures of isomers can be separated on the basis of thephysicochemical differences of the constituents, into the pure orsubstantially pure geometric or optical isomers, diastereomers,racemates, for example, by chromatography and/or fractionalcrystallization.

Any resulting racemates of final products or intermediates can beresolved into the optical antipodes by known methods, e.g., byseparation of the diastereomeric salts thereof, obtained with anoptically active acid or base, and liberating the optically activeacidic or basic compound. In particular, a basic moiety may thus beemployed to resolve the compounds of the present invention into theiroptical antipodes, e.g., by fractional crystallization of a salt formedwith an optically active acid, e.g., tartaric acid, dibenzoyl tartaricacid, diacetyl tartaric acid, di-O,O′-p-toluoyl tartaric acid, mandelicacid, malic acid or camphor-10-sulfonic acid. Racemic products can alsobe resolved by chiral chromatography, e.g., high pressure liquidchromatography (HPLC) using a chiral adsorbent.

Furthermore, the compounds of the present invention, including theirsalts, can also be obtained in the form of their hydrates, or includeother solvents used for their crystallization. The compounds of thepresent invention may inherently or by design form solvates withpharmaceutically acceptable solvents (including water); therefore, it isintended that the invention embrace both solvated and unsolvated forms.The term “solvate” refers to a molecular complex of a compound of thepresent invention (including salt or zwitterionic forms thereof) withone or more solvent molecules. Such solvent molecules are those commonlyused in the pharmaceutical art, which are known to be innocuous to therecipient, e.g., water, ethanol, and the like. The term “hydrate” refersto the complex where the solvent molecule is water.

The compounds of the present invention, including salts, hydrates andsolvates thereof, may inherently or by design form polymorphs.

As used herein, the terms “salt” or “salts” refers to an acid additionor base addition salt of a compound of the present invention. “Salts”include in particular “pharmaceutically acceptable salts”. The term“pharmaceutically acceptable salts” refers to salts that retain thebiological effectiveness and properties of the compounds of thisinvention and, which typically are not biologically or otherwiseundesirable. In many cases, the compounds of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or sulfate groups or groups similar thereto.

Pharmaceutically acceptable acid addition or exchange salts can beformed with inorganic acids and organic acids, e.g., acetate, aspartate,benzoate, besylate, bromide/hydrobromide, bicarbonate/carbonate,bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride,chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate,gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate,lactate, lactobionate, laurylsulfate, malate, maleate, malonate,mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate,nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate,propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate andtrifluoroacetate salts.

Inorganic acids or “Anionic Groups” that can be introduced or from whichsalts can be derived include, for example, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike.

Organic acids or “Anionic Groups” that can be introduced or from whichsalts can be derived include, for example, acetic acid, propionic acid,glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid,sulfosalicylic acid, and the like. Pharmaceutically acceptable baseaddition or exchange salts can be formed with inorganic and organicbases.

Inorganic bases or “Cationic Groups” that can be introduced or fromwhich salts can be derived include, for example, ammonium salts andmetals from columns I to XII of the periodic table. In certainembodiments, the salts are derived from the cationic groups sodium,potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper;particularly suitable salts include ammonium, potassium, sodium, calciumand magnesium salts.

Organic bases or “Cationic Groups” that can be introduced or from whichsalts can be derived include, for example, primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines, basic ion exchange resins, and thelike. Certain organic amines include isopropylamine, benzathine,cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazineand tromethamine.

The salts of the present invention can be synthesized from a basic oracidic moiety, by conventional chemical methods. Generally, such saltscan be prepared by reacting free acid forms of these compounds with astoichiometric amount of the appropriate base (such as Na, Ca, Mg, or Khydroxide, carbonate, bicarbonate or the like), or by reacting free baseforms of these compounds with a stoichiometric amount of the appropriateacid. Preferably, a salt of a compound of the invention, such as anammonium salt, may be subjected to an ion exchange resin in its alkalimetal or alkaline earth metal form to promote a counterion exchange.Acid addition or exchange salts of compounds of the present inventionare obtained in customary manner, e.g. by treating the compounds with anacid or a suitable anion exchange reagent. Zwitterions or internal saltsof compounds of the present invention containing acid and basicsalt-forming groups, e.g. a free sulfate group and a free amino group,may be formed, e.g. by the neutralization of salts, such as acidaddition salts, to the isoelectric point, e.g. with weak bases, or bytreatment with ion exchangers. Such reactions are typically carried outin water or in an organic solvent, or in a mixture of the two.Generally, use of non-aqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile is desirable, where practicable. Additionalsuitable salts can be found, e.g., in “Remington's PharmaceuticalSciences”, 20th ed., Mack Publishing Company, Easton, Pa., (1985); andin “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” byStahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds of the presentinvention. Isotopically labeled compounds have structures depicted bythe formulas given herein except that one or more atoms are replaced byan atom having a selected atomic mass or mass number. Examples ofisotopes that can be incorporated into compounds of the inventioninclude isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous,fluorine, and chlorine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F ³¹P,³²P, ³⁵S, ³⁶Cl, ¹²⁵I respectively. The invention includes variousisotopically labeled compounds of the present invention, for examplethose into which radioactive isotopes, such as ³H and ¹⁴C, or those intowhich non-radioactive isotopes, such as ²H and ¹³C are present. Suchisotopically labelled compounds are useful in metabolic studies (with¹⁴C), reaction kinetic studies (with, for example ²H or ³H), detectionor imaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. In particular, an ¹⁸F labeled compound of the presentinvention may be particularly desirable for PET or SPECT studies.Isotopically-labeled compounds of the present invention can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described in the accompanying Examplesand Preparations using an appropriate isotopically-labeled reagent inplace of the non-labeled reagent previously employed.

Further, substitution with heavier isotopes, particularly deuterium(i.e., ²H or D) may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example increased in vivo half-life orreduced dosage requirements or an improvement in therapeutic index. Itis understood that deuterium in this context is regarded as asubstituent of a compound of the present invention. The concentration ofsuch a heavier isotope, specifically deuterium, may be defined by theisotopic enrichment factor. The term “isotopic enrichment factor” asused herein means the ratio between the isotopic abundance and thenatural abundance of a specified isotope. If a substituent in a compoundof this invention is denoted deuterium, such compound has an isotopicenrichment factor for each designated deuterium atom of at least 3500(52.5% deuterium incorporation at each designated deuterium atom), atleast 4000 (60% deuterium incorporation), at least 4500 (67.5% deuteriumincorporation), at least 5000 (75% deuterium incorporation), at least5500 (82.5% deuterium incorporation), at least 6000 (90% deuteriumincorporation), at least 6333.3 (95% deuterium incorporation), at least6466.7 (97% deuterium incorporation), at least 6600 (99% deuteriumincorporation), or at least 6633.3 (99.5% deuterium incorporation).

Pharmaceutically acceptable solvates in accordance with the inventioninclude those wherein the solvent of crystallization may be isotopicallysubstituted, e.g. D₂O, d₆-acetone, d₆-DMSO.

Compounds of the present invention that contain groups capable of actingas donors and/or acceptors for hydrogen bonds may be capable of formingco-crystals with suitable co-crystal formers. These co-crystals may beprepared from compounds of the present invention by known co-crystalforming procedures. Such procedures include grinding, heating,co-subliming, co-melting, or contacting in solution compounds of thepresent invention with the co-crystal former under crystallizationconditions and isolating co-crystals thereby formed. Suitable co-crystalformers include those described in WO 2004/078163. Hence the inventionfurther provides co-crystals comprising a compound of the presentinvention.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

The present invention provides novel compounds, pharmaceuticalformulations including the compounds, and methods of treatingGram-negative bacterial infections. Particularly, the compounds aresuitable for use to treat infections caused by Burkholderia,Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella,Pseudomonas, Proteus, Salmonella, Serratia, Acinetobacter, Bacteroides,Campylobacter, Neisseria, or Stenotrophomonas bacteria, includingspecies named herein.

Substitution with heavier isotopes such as deuterium, i.e. ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances. Forexample, deuterium substitution at non-exchangeable hydrocarbon bonds(e.g., C—H) may retard epimerization and/or metabolic oxidation in vivo.

Isotopically-labeled compounds of the invention, i.e. compounds offormula (A), can generally be prepared by conventional techniques knownto those skilled in the art or by processes analogous to those describedin the accompanying Examples and Preparations Sections using anappropriate isotopically-labeled reagent in place of the non-labeledreagent previously.

In still another aspect, the invention provides a method for treating asubject with a bacterial infection, the method comprising the step ofadministering to the subject in need thereof an antibacteriallyeffective amount of a compound of the invention, e.g., a compound ofFormula (A) or salt thereof with a pharmaceutically acceptable carrier,in combination with a beta-lactam antibiotic. Suitable beta-lactamantibiotics for use in these methods include, but are not limited to,penicillins such as penicillin G, penicillin V, methicillin, oxacillin,cloxacillin, dicloxacillin, nafcillin, ampicillin, amoxicillin,carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin,temocillin, cephalosporins such as cepalothin, cephapirin, cephradine,cephaloridine, cefazolin, cefamandole, cefuroxime, cephalexin,cefprozil, cefaclor, loracarbef, cefoxitin, cefinetazole, cefotaxime,ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime,cefpodoxime, ceftibuten, cefdinir, cefpirome, cefepime, ceftolozane;carbapenems such as doripenem, imipenem, meropenem, panipenem, biapenem;and monobactams such as aztreonam, and beta-lactam 5, which is disclosedherein.

An “effective amount” of a compound of the invention is an amount thatsubstantially potentiates the activity of a beta-lactam antibiotic usedin combination with the compound of the invention, such as an amountthat causes the antibiotic to be at least four times more active againsta target bacterium, i.e. an amount that lowers the minimum inhibitoryconcentration (or “minimal inhibitory concentration”, “MIC”) for thetarget bacterium by at least 4× and preferably by at least 8×.

An “effective amount” of a combination of a BLI plus beta-lactamantibiotic as used herein refers to an amount effective to treat abacterial infection in a subject, typically a human subject. Theeffective amount depends upon the sensitivity of the infecting bacteriumto the chosen antibiotic and on the degree of potentiation provided bythe BLI used in the combination. The skilled person can determine aneffective amount of such combinations based on parameters of the subjectto be treated, the infecting bacterium, and the combination to be used,which may include determining the MIC for the particular combination onthe targeted bacterium. Typically, the bacterium to be treated is onethat is resistant to at least some beta-lactam antibiotics because thebacterium expresses a beta-lactamase activity.

The compounds of the invention also are useful in the treatment ofpatients suffering from or susceptible to skin infections, pneumonia,sepsis, cystic fibrosis, wound, complicated diabetic foot, complicationintra abdominal infections or complicated urinary tract infections andsexually transmitted diseases caused by Gram-negative or Gram-positivepathogens. The compounds of the invention also are useful in theconditions that are caused by a species of Citrobacter, Enterobacter,Escherichia, Klebsiella, Morganella, Proteus, Salmonella, Serratia,Pseudomonas, Acinetobacter, Bacteroides, Burkholderia, Campylobacter,Neisseria, or Stenotrophomonas. In particular, a bacterial infectioncaused by a species of Citrobacter, Enterobacter, Escherichia,Klebsiella, Morganella, Proteus, Salmonella, Serratia, Pseudomonas, orAcinetobacter is treatable by methods herein. Particular bacterialspecies for such treatment include Citrobacter freundii, Citrobacterkoseri, Enterobacter cloacae, Enterobacter faecalis, Enterobacterfaecium, Escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca,Morganella morganii, Proteus mirabilis, Salmonella species, Serratiamarcescens, Pseudomonas aeruginosa, and Acinetobacter baumannii, as wellas Bacteroides fragilis, Burkholderia cepacia, Campylobacter jejuni,Neisseria gonorrhoeae, and Stenotrophomonas maltophilia.

By the term “combination”, is meant either a fixed combination in onedosage unit form, or a kit or instructions for the combinedadministration where a compound of the present invention and acombination beta-lactam antibiotic partner may be administeredindependently or together, at the same time or separately within timeintervals that especially allow that the combination partners show acooperative, e.g., synergistic, effect, or any combination thereof.

An embodiment of the present invention provides compounds of the presentinvention in a pharmaceutical combination with a beta-lactam antibioticand a third therapeutic agent. In some embodiments, the thirdtherapeutic agent is an additional antibacterial agent or an additionalbeta-lactamase inhibitor. In some embodiments, the combination includesat least one other antibacterial agent, which may be another beta-lactamantibiotic or another antibacterial agent selected from the classesdescribed below. Non-limiting examples of additional antibacterialagents for use in pharmaceutical combinations of the invention may beselected from the following groups:

(1) Macrolides or ketolides such as erythromycin, azithromycin,clarithromycin, and telithromycin;

(2) Beta-lactam antibiotics including penicillin such as penicillin G,penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin,nafcillin, ampicillin, amoxicillin, carbenicillin, ticarcillin,mezlocillin, piperacillin, azlocillin, temocillin, cephalosporin such ascepalothin, cephapirin, cephradine, cephaloridine, cefazolin,cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor, loracarbef,cefoxitin, cefinetazole, cefotaxime, ceftizoxime, ceftriaxone,cefoperazone, ceftazidime, cefixime, cefpodoxime, ceftibuten, cefdinir,cefpirome, cefepime, ceftolozane and carbapenems such as doripenem,imipenem, meropenem, panipenem, and monobactams such as aztreonam, andbeta-lactam 5 herein;

(3) Glycopeptides such as vancomycin and teicoplanin;

(4) Quinolones such as nalidixic acid, oxolinic acid, norfloxacin,pefloxacin, enoxacin, ofloxacin, levofloxacin, ciprofloxacin,temafloxacin, lomefloxacin, fleroxacin, grepafloxacin, sparfloxacin,trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, sitafloxacin,ganefloxacin, gemifloxacin, delafloxacin and pazufloxacin;

(5) Antibacterial sulfonamides and antibacterial sulphanilamides,including para-aminobenzoic acid, sulfadiazine, sulfisoxazole,sulfamethoxazole and sulfathalidine;

(6) Aminoglycosides such as streptomycin, neomycin, kanamycin,paromycin, gentamicin, tobramycin, amikacin, netilmicin, spectinomycin,sisomicin, dibekalin, plazomicin and isepamicin;

(7) Tetracyclines such as tetracycline, chlortetracycline,demeclocycline, minocycline, oxytetracycline, methacycline, doxycycline,tigecycline and eravacyclin;

(8) Rifamycins such as rifampicin (also called rifampin), rifapentine,rifabutin, bezoxazinorifamycin and rifaximin;

(9) Lincosamides such as lincomycin and clindamycin;

(10) Streptogramins such as quinupristin and daflopristin;

(11) Oxazolidinones such as linezolid or tedizolid;

(12) Polymyxin, colistin and colymycin;

(13) Trimethoprim and bacitracin; and

(14) Efflux pump inhibitors

(15) Beta-lactamase inhibitors, such as metallo beta-lactamaseinhibitors.

The beta-lactam or second antibacterial agent may be administered incombination with the compounds of the present inventions wherein thebeta-lactam or second antibacterial agent is administered prior to,simultaneously, or after the compound or compounds of the presentinvention. When simultaneous administration of a compound of theinvention with a second or third agent is desired and the route ofadministration is the same, then a compound of the invention may beformulated with a second or third agent into the same dosage form. Anexample of a dosage form containing a compound of the invention and asecond or third agent is an intravenous administration. An alternativeexample is an intramuscular administration of a solution comprising acompound of the invention and a second or third agent.

The compounds and compositions described herein can be used oradministered in combination with a beta-lactam and one or moretherapeutic agents that act as immunomodulators, e.g., an activator of acostimulatory molecule, or an inhibitor of an immune-inhibitorymolecule, or a vaccine. The Programmed Death 1 (PD-1) protein is aninhibitory member of the extended CD28/CTLA4 family of T cell regulators(Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al.(2003) J. Immunol. 170:711-8). PD-1 is expressed on activated B cells, Tcells, and monocytes. PD-1 is an immune-inhibitory protein thatnegatively regulates TCR signals (Ishida, Y. et al. (1992) EMBO J.11:3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother.56(5):739-745), and is up-regulated in chronic infections. Theinteraction between PD-1 and PD-L1 can act as an immune checkpoint,which can lead to, e.g., a decrease in infiltrating lymphocytes, adecrease in T-cell receptor mediated proliferation, and/or immuneevasion by cancerous or infected cells (Dong et al. (2003) J. Mol. Med.81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314;Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppressioncan be reversed by inhibiting the local interaction of PD-1 with PD-L1or PD-L2; the effect is additive when the interaction of PD-1 with PD-L2is blocked as well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).Immunomodulation can be achieved by binding to either theimmune-inhibitory protein (e.g., PD-1) or to binding proteins thatmodulate the inhibitory protein (e.g., PD-L1, PD-L2).

In one embodiment, the combination therapies of the invention include animmunomodulator that is an inhibitor or antagonist of an inhibitorymolecule of an immune checkpoint molecule. In another embodiment theimmunomodulator binds to a protein that naturally inhibits theimmuno-inhibitory checkpoint molecule. When used in combination withantibacterial compounds, these immunomodulators can enhance theantimicrobial response, and thus enhance efficacy relative to treatmentwith the antibacterial compound alone.

The term “immune checkpoints” refers to a group of molecules on the cellsurface of CD4 and CD8 T cells. These molecules can effectively serve as“brakes” to down-modulate or inhibit an adaptive immune response. Immunecheckpoint molecules include, but are not limited to, Programmed Death 1(PD-1), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), B7H1, B7H4, OX-40,CD137, CD40, and LAG3, which directly inhibit immune cells.Immunotherapeutic agents which can act as immune checkpoint inhibitorsuseful in the methods of the present invention, include, but are notlimited to, inhibitors of PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA,TIGIT, LAIR1, CD160, 2B4 and/or TGFR beta. Inhibition of an inhibitorymolecule can be performed by inhibition at the DNA, RNA or proteinlevel. In some embodiments, an inhibitory nucleic acid (e.g., a dsRNA,siRNA or shRNA), can be used to inhibit expression of an inhibitorymolecule. In other embodiments, the inhibitor of an inhibitory signal isa polypeptide, e.g., a soluble ligand, or an antibody or antigen-bindingfragment thereof, that binds to the inhibitory molecule.

By “in combination with,” it is not intended to imply that the therapyor the therapeutic agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope described herein. The immunomodulator can beadministered concurrently with, prior to, or subsequent to, one or morecompounds of the invention and the beta-lactam partner, and optionallyone or more additional therapies or therapeutic agents. The therapeuticagents in the combination can be administered in any order. In general,each agent will be administered at a dose and/or on a time scheduledetermined for that agent. It will further be appreciated that thetherapeutic agents utilized in this combination may be administeredtogether in a single composition or administered separately in differentcompositions. In general, it is expected that each of the therapeuticagents utilized in combination be utilized at levels that do not exceedthe levels at which they are utilized individually. In some embodiments,the levels utilized in combination will be lower than those utilizedindividually.

In certain embodiments, the beta-inhibitor described herein areadministered in combination with a beta-lactam and one or moreimmunomodulators that are inhibitors of PD-1, PD-L1 and/or PD-L2. Eachsuch inhibitor may be an antibody, an antigen binding fragment thereof,an immunoadhesin, a fusion protein, or an oligopeptide. Examples of suchimmunomodulators are known in the art.

In some embodiments, the immunomodulator is an anti-PD-1 antibody chosenfrom MDX-1106, Merck 3475 or CT-011.

In some embodiments, the immunomodulator is an immunoadhesin (e.g., animmunoadhesin comprising an extracellular or PD-1 binding portion ofPD-LI or PD-L2 fused to a constant region (e.g., an Fc region of animmunoglobulin sequence).

In some embodiments, the immunomodulator is a PD-1 inhibitor such asAMP-224.

In some embodiments, the immunomodulator is a PD-LI inhibitor such asanti-PD-LI antibody.

In some embodiments, the immunomodulator is an anti-PD-LI bindingantagonist chosen from YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C,or MDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-LIantibody described in WO2007/005874. Antibody YW243.55.S70 is ananti-PD-LI described in WO 2010/077634.

In some embodiments, the immunomodulator is nivolumab (CAS RegistryNumber: 946414-94-4). Alternative names for nivolumab include MDX-1106,MDX-1106-04, ONO-4538, or BMS-936558. Nivolumab is a fully human IgG4monoclonal antibody which specifically blocks PD-1. Nivolumab (clone5C4) and other human monoclonal antibodies that specifically bind toPD-1 are disclosed in U.S. Pat. No. 8,008,449, EP2161336 andWO2006/121168.

In some embodiments, the immunomodulator is an anti-PD-1 antibodyPembrolizumab. Pembrolizumab (also referred to as Lambrolizumab,MK-3475, MK03475, SCH-900475 or KEYTRUDA®; Merck) is a humanized IgG4monoclonal antibody that binds to PD-1. Pembrolizumab and otherhumanized anti-PD-1 antibodies are disclosed in Hamid, O. et al. (2013)New England Journal of Medicine 369 (2): 134-44, U.S. Pat. No.8,354,509, WO2009/114335, and WO2013/079174.

In some embodiments, the immunomodulator is Pidilizumab (CT-011; CureTech), a humanized IgG1k monoclonal antibody that binds to PD1.Pidilizumab and other humanized anti-PD-1 monoclonal antibodies aredisclosed in WO2009/101611.

Other anti-PD1 antibodies useful as immunomodulators for use in themethods disclosed herein include AMP 514 (Amplimmune), and anti-PD1antibodies disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/orUS 20120114649. In some embodiments, the anti-PD-L1 antibody isMSB0010718C. MSB0010718C (also referred to as A09-246-2; Merck Serono)is a monoclonal antibody that binds to PD-L1.

In some embodiments, the immunomodulator is MDPL3280A (Genentech/Roche),a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1.MDPL3280A and other human monoclonal antibodies to PD-L1 are disclosedin U.S. Pat. No. 7,943,743 and U.S Publication No.: 20120039906. Otheranti-PD-L1 binding agents useful as immunomodulators for methods of theinvention include YW243.55.S70 (see WO2010/077634), MDX-1105 (alsoreferred to as BMS-936559), and anti-PD-L1 binding agents disclosed inWO2007/005874.

In some embodiments, the immunomodulator is AMP-224 (B7-DCIg;Amplimmune; e.g., disclosed in WO2010/027827 and WO2011/066342), is aPD-L2 Fc fusion soluble receptor that blocks the interaction between PD1and B7-H1.

In some embodiments, the immunomodulator is an anti-LAG-3 antibody suchas BMS-986016. BMS-986016 (also referred to as BMS986016) is amonoclonal antibody that binds to LAG-3. BMS-986016 and other humanizedanti-LAG-3 antibodies are disclosed in US 2011/0150892, WO2010/019570,and WO2014/008218

In certain embodiments, the combination therapies disclosed hereininclude a modulator of a costimulatory molecule or an inhibitorymolecule, e.g., a co-inhibitory ligand or receptor.

In one embodiment, the costimulatory modulator, e.g., agonist, of acostimulatory molecule is chosen from an agonist (e.g., an agonisticantibody or antigen-binding fragment thereof, or soluble fusion) ofOX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB(CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7,NKp80, CD160, B7-H3 or CD83 ligand.

In another embodiment, the combination therapies disclosed hereininclude an immunomodulator that is a costimulatory molecule, e.g., anagonist associated with a positive signal that includes a costimulatorydomain of CD28, CD27, ICOS and/or GITR.

Exemplary GITR agonists include, e.g., GITR fusion proteins andanti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as, aGITR fusion protein described in U.S. Pat. No. 6,111,090, EuropeanPatent No.: 090505B1, U.S. Pat. No. 8,586,023, PCT Publication Nos.: WO2010/003118 and 2011/090754, or an anti-GITR antibody described, e.g.,in U.S. Pat. No. 7,025,962, European Patent No.: 1947183B1, U.S. Pat.Nos. 7,812,135, 8,388,967, 8,591,886, European Patent No.: EP 1866339,PCT Publication No.: WO 2011/028683, PCT Publication No.: WO2013/039954, PCT Publication No.: WO2005/007190, PCT Publication No.: WO2007/133822, PCT Publication No.: WO2005/055808, PCT Publication No.: WO99/40196, PCT Publication No.: WO 2001/03720, PCT Publication No.:WO99/20758, PCT Publication No.: WO2006/083289, PCT Publication No.: WO2005/115451, U.S. Pat. No. 7,618,632, and PCT Publication No.: WO2011/051726.

In one embodiment, the immunomodulator used is a soluble ligand (e.g., aCTLA-4-Ig), or an antibody or antibody fragment that binds to PD-L1,PD-L2 or CTLA4. For example, the anti-PD-1 antibody molecule can beadministered in combination with an anti-CTLA-4 antibody, e.g.,ipilimumab, for example. Exemplary anti-CTLA4 antibodies includeTremelimumab (IgG2 monoclonal antibody available from Pfizer, formerlyknown as ticilimumab, CP-675,206); and Ipilimumab (CTLA-4 antibody, alsoknown as MDX-010, CAS No. 477202-00-9).

In one embodiment, an anti-PD-1 antibody molecule is administered aftertreatment with a compound of the invention as described herein.

In another embodiment, an anti-PD-1 or PD-L1 antibody molecule isadministered in combination with an anti-LAG-3 antibody or anantigen-binding fragment thereof. In another embodiment, the anti-PD-1or PD-L1 antibody molecule is administered in combination with ananti-TIM-3 antibody or antigen-binding fragment thereof. In yet otherembodiments, the anti-PD-1 or PD-L1 antibody molecule is administered incombination with an anti-LAG-3 antibody and an anti-TIM-3 antibody, orantigen-binding fragments thereof. The combination of antibodies recitedherein can be administered separately, e.g., as separate antibodies, orlinked, e.g., as a bispecific or trispecific antibody molecule. In oneembodiment, a bispecific antibody that includes an anti-PD-1 or PD-L1antibody molecule and an anti-TIM-3 or anti-LAG-3 antibody, orantigen-binding fragment thereof, is administered. In certainembodiments, the combination of antibodies recited herein is used totreat a cancer, e.g., a cancer as described herein (e.g., a solidtumor). The efficacy of the aforesaid combinations can be tested inanimal models known in the art. For example, the animal models to testthe synergistic effect of anti-PD-1 and anti-LAG-3 are described, e.g.,in Woo et al. (2012) Cancer Res. 72(4):917-27).

Exemplary immunomodulators that can be used in the combination therapiesinclude, but are not limited to, e.g., afutuzumab (available fromRoche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®);thalidomide (Thalomid®), actimid (CC4047); and cytokines, e.g., IL-21 orIRX-2 (mixture of human cytokines including interleukin 1, interleukin2, and interferon γ, CAS 951209-71-5, available from IRX Therapeutics).

Exemplary doses of such immunomodulators that can be used in combinationwith the antibacterial compounds of the invention include a dose ofanti-PD-1 antibody molecule of about 1 to 10 mg/kg, e.g., 3 mg/kg, and adose of an anti-CTLA-4 antibody, e.g., ipilimumab, of about 3 mg/kg.

Examples of embodiments of the methods of using the compounds of theinvention in combination with a beta-lactam antibiotic and animmunomodulator include these:

i. A method to treat a bacterial infection in a subject, comprisingadministering to the subject a compound of Formula (A) as describedherein, and an immunomodulator.

ii. The method of embodiment i, wherein the immunomodulator is anactivator of a costimulatory molecule or an inhibitor of an immunecheckpoint molecule.

iii. The method of either of embodiments i and ii, wherein the activatorof the costimulatory molecule is an agonist of one or more of OX40, CD2,CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137),GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160,B7-H3 and CD83 ligand.

iv. The method of any of embodiments i-iii above, wherein the inhibitorof the immune checkpoint molecule is chosen from PD-1, PD-L1, PD-L2,CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.

v. The method of any of any of embodiments i-iii, wherein the inhibitorof the immune checkpoint molecule is chosen from an inhibitor of PD-1,PD-L1, LAG-3, TIM-3 or CTLA4, or any combination thereof.

vi. The method of any of embodiments i-v, wherein the inhibitor of theimmune checkpoint molecule is a soluble ligand or an antibody orantigen-binding fragment thereof, that binds to the immune checkpointmolecule.

vii. The method of any of embodiments i-vi, wherein the antibody orantigen-binding fragment thereof is from an IgG1 or IgG4 (e.g., humanIgG1 or IgG4).

viii. The method of any of embodiments i-vii, wherein the antibody orantigen-binding fragment thereof is altered, e.g., mutated, to increaseor decrease one or more of: Fc receptor binding, antibody glycosylation,the number of cysteine residues, effector cell function, or complementfunction.

ix. The method of any of embodiments i-viii, wherein the antibodymolecule is a bispecific or multispecific antibody molecule that has afirst binding specificity to PD-1 or PD-L1 and a second bindingspecificity to TIM-3, LAG-3, or PD-L2.

x. The method of any of embodiments i-ix, wherein the immunomodulator isan anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab orPidilizumab.

xi. The method of any of embodiments i-x, wherein the immunomodulator isan anti-PD-L1 antibody chosen from YW243.55.S70, MPDL3280A, MEDI-4736,MSB-0010718C, or MDX-1105.

xii. The method of any of embodiments i-x, wherein the immunomodulatoris an anti-LAG-3 antibody molecule.

xiii. The method of embodiment xii, wherein the anti-LAG-3 antibodymolecule is BMS-986016,

xiv. The method of any of embodiments i-x, wherein the immunomodulatoris an anti-PD-1 antibody molecule administered by injection (e.g.,subcutaneously or intravenously) at a dose of about 1 to 30 mg/kg, e.g.,about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about3 mg/kg., e.g., once a week to once every 2, 3, or 4 weeks.

xv. The method of embodiment xiv, wherein the anti-PD-1 antibodymolecule is administered at a dose from about 10 to 20 mg/kg every otherweek.

xvi. The method of embodiment xv, wherein the anti-PD-1 antibodymolecule, e.g., nivolumab, is administered intravenously at a dose fromabout 1 mg/kg to 3 mg/kg, e.g., about 1 mg/kg, 2 mg/kg or 3 mg/kg, everytwo weeks.

xvii. The method of embodiment xv, wherein the anti-PD-1 antibodymolecule, e.g., nivolumab, is administered intravenously at a dose ofabout 2 mg/kg at 3-week intervals.

The language “effective amount” of the compound is that amount necessaryor sufficient to enhance the efficacy of a beta-lactam antibiotic usedto treat or prevent a bacterial infection and/or a disease or conditiondescribed herein. In an example, an effective amount of the compound isan amount sufficient to treat bacterial infection in a subject, whendosed together with a beta-lactam. In another example, an effectiveamount of the compound is an amount sufficient to treat a bacterialinfection, when dosed in combination with a beta-lactam antibiotic,caused by, but not limited to species of Enterobacteriaceae and the likein a subject. The effective amount can vary depending on such factors asthe size and weight of the subject, the type of illness, thecharacteristics of the bacterial pathogen causing the illness (forexample the type and level of beta-lactamase production) or theparticular compound of the invention, as well as the beta-lactamantibiotic to be used along with the compound of the invention. Forexample, the choice of the compound of the invention can affect whatconstitutes an “effective amount.” One of ordinary skill in the artwould be able to study the factors contained herein and make thedetermination regarding the effective amount of the compounds of theinvention without undue experimentation.

The regimen of administration can affect what constitutes an effectiveamount. The compound of the invention can be administered to the subjecteither prior to or after the onset of a bacterial infection. Typically,the compound is administered to a subject diagnosed as having abacterial infection and in need of treatment therefore. Further, severaldivided dosages, as well as staggered dosages, can be administered every6 hours, every 8 hours, every 12 hours or daily or sequentially, or thedose can be continuously infused, or can be a bolus injection. Further,the dosages of the compound(s) of the invention can be proportionallyincreased or decreased as indicated by the exigencies of the therapeuticor prophylactic situation. Typically, the compound of the inventionwould be administered over a course of at least 5 days, more commonly atleast 7 days or at least 10 days or at least 14 days, through 3 or 4infusions per day (every 6 or 8 hours).

Compounds of the invention may be used in the treatment of states,disorders or diseases as described herein, or for the manufacture ofpharmaceutical compositions for use in the treatment of these diseases.The invention provides methods of use of compounds of the presentinvention in the treatment of these diseases or pharmaceuticalpreparations having compounds of the present invention for the treatmentof these diseases.

The language “pharmaceutical composition” includes preparations suitablefor administration to mammals, e.g., humans. When the compounds of thepresent invention are administered as pharmaceuticals to mammals, e.g.,humans, they can be given per se or as a pharmaceutical compositioncontaining, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) ofactive ingredient in combination with a pharmaceutically acceptablecarrier.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations. In someembodiments, a pharmaceutically acceptable carrier is sterilized beforecombination with the compound of the invention.

In some embodiments, the pharmaceutical composition of the inventioncomprises a compound of any of the numbered embodiments and at least onepharmaceutically acceptable carrier or excipient. In certainembodiments, the pharmaceutical composition of the invention comprises acompound of any of the numbered embodiments and at least twopharmaceutically acceptable carriers or excipients.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as preservatives andantioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, α-tocopherol, and the like; and metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, inhalation, topical, transdermal, buccal, sublingual, rectal,vaginal and/or parenteral administration. Typically, compounds of theinvention would be administered intravenously, in the form of a solutionthat is often isotonic, such as a saline or glucose solution. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof active ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound that produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc., administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Intravenous administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including intramuscularinjection, orally, nasally, inhaled as by, for example, a spray,rectally, intravaginally, parenterally, intracisternally and topically,as by powders, ointments or drops, including buccally and sublingually.In some embodiments, the compound of the invention is administered byinjection or infusion, often by infusion, and it may be co-administeredwith a beta-lactam antibiotic. The beta-lactam antibiotic may beadministered by any appropriate route; in some embodiments, thebeta-lactam antibiotic is administered orally, and in other embodimentsthe beta-lactam antibiotic is administered by injection or by infusion.When the compound of the invention is co-administered with a beta-lactamantibiotic and both are administered by the same route, they mayoptionally be admixed for administration by injection or by infusion, orthey may be separately administered provided the beta-lactamaseinhibitor is present systemically in the treated subject along with thebeta-lactam antibiotic so potentiation can occur.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the salt thereof, the route of administration,the time of administration, the rate of excretion of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, the genus, speciesand strain of bacterial pathogen causing the infection and like factorswell known in the medical arts.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous and subcutaneousdoses of the compounds of this invention for a patient, when used incombination with a beta-lactam for the indicated antibacterial effects,will range from about 2 to about 100 mg per kilogram of body weight perday, more preferably from about 5 to about 100 mg per kg per day, andstill more preferably from about 10 to about 50 mg per kg per day. Aneffective amount is that amount treats a bacterial infection, when dosedin combination with a beta-lactam antibiotic.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms, or as continuous infusion.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

The compounds as defined in embodiments may be synthesized by thegeneral synthetic routes below, specific examples of which are describedin more detail in the Examples.

General Synthetic Schemes

One method for synthesizing compounds of Formula (I) is depicted in thefollowing reaction schemes. Scheme A illustrates functionalization ofthe known diazabicyclooctane skeleton in protected form to introduce anaminoalkyl group, as described in the working examples. Scheme Billustrates formation of the fused lactam ring, which is alsoillustrated by the Examples. Scheme C illustrates how the lactam couldreadily be N-alkylated to introduce an optionally-substituted alkylgroup.

Examples of sulfonylating agents include, but are not limited tosulfurtrioxide pyridine complex, and the like.

Examples of bases include, but are not limited to pyridine and the like.

EXAMPLES

The invention is further illustrated by the following examples, whichshould not be construed as limiting. The assays used throughout theExamples are accepted. Demonstration of efficacy in these assays ispredictive of efficacy in subjects.

General Conditions

Mass spectra were acquired on LC-MS, SFC-MS, or GC-MS systems usingelectrospray, chemical and electron impact ionization methods from arange of instruments of the following configurations: Waters ACQUITYUPLC system and equipped with a ZQ 2000 or SQD MS system where (M+1)refers to the protonated molecular ion of the chemical species, (M+)refers to the unprotonated quaternary ammonium cation and (M−1) refersto the deprotonated molecular ion of the chemical species.

NMR spectra were run on a Bruker BioSpin 600 MHz, Bruker AVANCE 500 MHzor Varian 400 MHz NMR spectrometers using ICON-NMR, under TopSpinprogram control. Spectra were measured at 298K, unless indicatedotherwise, and were referenced relative to the solvent resonance.

Instrumentation

MS Methods: Method 2m_acidic: Column Kinetex C18 50 × 2.1 mm, 2.6 μmColumn Temperature 50° C. Eluents A: H₂O, B: acetonitrile, bothcontaining 0.1% TFA Flow Rate 1.2 mL/min Gradient 2% to 88% B in 1.30min, 0.15 min 95% B Method 2m_acidic_polar: Column Kinetex C18 50 × 2.1mm, 2.6 μm Column Temperature 50° C. Eluents A: H₂O, B: acetonitrile,both containing 0.1% TFA Flow Rate 1.2 mL/min Gradient 1% to 30% B in1.30 min, 0.15 min 98% B Method T3_3m_polar: Column T3 C18 50 × 2.1 mm,2.6 μm Column Temperature 50° C. Eluents A: H₂O, B: acetonitrile, bothcontaining 0.1% TFA Flow Rate 1.2 mL/min Gradient 100% A for 1.1 min,30% B in 1.20 min, 95% B in 0.7 min

Method LCMS 2 MIN_REACTION_MONITORING:

Column Acquity UPLC HSS T3 50 × 2.1 mm, 1.8 μm Column 60° C. TemperatureEluents A: H₂O, B: acetonitrile, both containing 0.05% TFA Flow Rate 1.0mL/min Gradient 5% to 98% B in 1.4 min UV detection TAC (210-450 nm)

Method LCMS 2 MIN_FINAL_ANALYSIS:

Column Acquity UPLC HSS T3 50 × 2.1 mm, 1.8 μm Column 60° C. TemperatureEluents A: H₂O (0.05% FA + 3.75 mM AA, B: acetonitrile (0.04% FA) FlowRate 1.0 mL/min Gradient 5% to 98% B in 1.4 min UV detection TAC(210-450 nm)

Method LCMS 2 MIN_Polar:

Column Acquity UPLC HSS T3 50 × 2.1 mm, 1.8 μm Column 60° C. TemperatureEluents A: H₂O (0.05% FA + 3.75 mM AA, B: acetonitrile (0.04% FA) FlowRate 1.0 mL/min Gradient concave from 1% to 98% B in 1.4 min UVdetection TAC (210-450 nm)

Method HPLC_CHIRAL:

Column Chiralpak IC KK025 250 × 4.6 mm, 5 μm Column Temperature rtEluents heptane/EtOH/diethylamine 92:8:0.05 Flow Rate 1.0 mL/min UVdetection 220 nm

Abbreviations

-   -   AA ammonium acetate    -   ACN acetonitrile    -   app apparent    -   ATP adenosine 5′-triphosphate    -   BINAP racemic 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl    -   Boc tertiary butyl carboxy    -   br broad    -   br s broad singlet    -   BSA bovine serum albumin    -   d doublet    -   dd doublet of doublets    -   DCC dicyclohexylcarbodiimide    -   DCE 1,2-dichloroethane    -   DCM dichloromethane    -   DIAD diisopropylazodicarboxylate    -   DIPEA diisopropylethylamine    -   DMAP 4-(N,N-dimethylamino)pyridine    -   DME 1,4-dimethoxyethane    -   DMF N,N-dimethylformamide    -   DMSO dimethylsulfoxide    -   EDTA ethylenediamine tetraacetic acid    -   ESI electrospray ionization    -   EtOAc ethyl acetate    -   FA formic acid    -   g gram    -   h hour(s)    -   HATU        1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]py1ridinium        3-oxid hexafluorophosphate    -   HBTU        1-[bis(dimethylamino)methylene]-1H-benzotriazoliumhexafluorophosphate(1-)        3-oxide    -   HCl hydrochloric acid    -   HOBt 1-hydroxybenzotriazole    -   HPLC high performance liquid chromatography    -   LCMS liquid chromatography and mass spectrometry    -   LDA lithium diisopropylamide    -   MeOH methanol    -   MS mass spectrometry    -   m multiplet    -   mg milligram    -   MIC minimum or minimal inhibitory concentration    -   min minutes    -   mL milliliter    -   mmol millimole    -   m/z mass to charge ratio    -   NMR nuclear magnetic resonance    -   o/n overnight    -   p pentet    -   PdCl₂(dppf)-CH₂Cl₂        1,1′-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride        dichloromethane complex    -   ppm parts per million    -   PyBOP benzotriazol-1-yloxytripyrrolidinophosphonium        hexafluorophosphate    -   q quartet    -   rac racemic    -   rbf round bottom flask    -   rt room temperature    -   Rt retention time    -   s singlet    -   t triplet    -   TBME methyl tert-butyl ether    -   TFA trifluoroacetic acid    -   TFAA trifluoroacetic acid anhydride    -   THF tetrahydrofuran    -   Tris.HCl aminotris(hydroxymethyl)methane hydrochloride        Preparation of Intermediates

Intermediate A: Methyl(2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate.To a solution of(2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylicacid (5.0 g, 18.1 mmol), MeOH (880 μL, 21.7 mmol) and DMAP (44 mg, 0.36mmol) in DCM (50 mL) at 0° C. was added DCC (3.92 g, 19.0 mmol). After 2h at rt it was iluted with DCM and washed with water then brine. Theaqueous layers were extracted with DCM (2×) and the combined organiclayers were dried over Na₂SO₄, filtered then concentrated in vacuo. Thecrude residue was triturated with diethyl ether, filtered andconcentrated in vacuo. The crude filtrate was purified silica gelchromatography to afford the title compound (4.3 g, 82%). LCMSR_(t)=0.87 min, m/z=291.3 (M+1), Method 2 MIN_REACTION_MONITORING.

Intermediate B: Methyl(5R)-6-(benzyloxy)-7-oxo-2-(phenylselanyl)-1,6-diazabicyclo[3.2.1]octane-2-carboxylate.To a solution of diisopropylamine (29.6 ml, 210 mmol) in THF (800 mL) at−70° C. was added n-butyllithium (1.6 M in hexanes, 108 ml, 172 mmol)drop-wise over 10 minutes. After stirring for 50 minutes at −73° C. asolution of methyl(2S,5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate(43.5 g, 150 mmol) in THF (350 mL) was added drop-wise over 45 minutes.After stirring at −78° C. for 1.5 hours phenylselenyl chloride (57.4 g,300 mmol) in THF (260 mL) was added dropwise over 45 minutes. Afterstirring at −78° C. for 45 min it was allowed to warm to −10° C. over 60minutes and stirred for an additional hour, whereupon it was cooled to−30° C. and quenched with HCl (2 M, 50 mL) followed by addition ofmethanol (250 mL). The mixture was allowed to reach rt over 15 min thendiluted with TBME (1 L) and washed with brine:water (2:1, 2 L). Thephases were separated and the organic phase was washed brine (2 L). Theaqueous layers were extracted with TBME (2×500 mL). The combined organicphases were dried over Na₂SO₄, filtered and concentrated in vacuo. Thecrude residue was purified via silica gel chromatography, affording thetitle compound (23.70 g, 36%, 3:1 mix of diastereomers) as a brown oil.LCMS Rt=1.12/1.16 min, m/z=447.3 (M+1), Method 2MIN_REACTION_MONITORING;¹H NMR (600 MHz, CDCl₃, major diastereomer) δ 7.59 (d, J=7.9 Hz, 2H),7.42-7.30 (m, 8H), 5.00 (d, J=11.5 Hz, 1H), 4.88 (d, J=11.5 Hz, 1H),4.15 (d, J=11.7 Hz, 1H), 3.68 (s, 3H), 3.35 (s, 1H), 3.18 (d, J=11.8 Hz,1H), 2.44 (ddd, J=17.4, 11.8, 6.6 Hz, 1H), 2.07-2.01 (m, 1H), 1.91 (dd,J=16.6, 5.8 Hz, 1H), 1.72 (td, J=12.9, 6.0 Hz, 1H).

Intermediate C: Methyl(5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo[3.2.1]oct-2-ene-2-carboxylate.To a solution of Intermediate B in THF:water (20:1, 22 mL) at 0° C. wasadded H₂O₂ (30% aq, 0.8 mL, 7.83 mmol) and AcOH (0.55 mL, 9.61 mmol).After stirring for 1 hour at 0° C. it was diluted with EtOAc andpotassium sulfite (5% aq) was added. Upon destruction of all peroxides(KJ-starke-test), the phases were separated and the organic layer waswashed with brine. The aqueous layers were extracted with EtOAc and thecombined organic layers were washed with NaHCO₃ (5% aq), brine, driedover Na₂SO₄, filtered and concentrated in vacuo. The crude residue waspurified via silica gel chromatography to afford the title compound (419mg, 81%). LCMS: R_(t)=0.88 min, m/z=288.1 (M+1), Method2MIN_FINAL_ANALYSIS. ¹H NMR (600 MHz, DMSO-d₆) δ 7.45-7.41 (m, 2H),7.40-7.33 (m, 3H), 6.88-6.86 (m, 1H), 4.89 (s, 2H), 3.96 (br s, 1H),3.67 (s, 3H), 3.35-3.28 (m, 1H), 2.82 (d, J=11.0 Hz, 1H), 2.58-2.52 (m,1H), 2.38 (s, 1H), 2.34 (s, 1H).

Intermediate D: N-benzyl-N-(tert-butoxycarbonyl)glycine. To a suspensionof N-benzylglycine (24.3 g, 147 mmol) in THF:water (1:1, 500 mL) wasadded Boc-anhydride (33.7 g, 154 mmol). After 6.5 h the mixture wasdiluted with TBME (250 mL) and citric acid (33 g) was added until pH=4.After 10 min of stirring, the phases were separated and the organicphase was washed with brine (250 ml). The aqueous layer was washed withTBME (2×100 ml) and the combined organic phases were dried over Na₂SO₄,filtered then concentrated in vacuo (45° C.), affording the titlecompound (40.70 g) as a colorless oil, which began to crystallize uponstanding. HPLC: 99.7% by UV, LCMS: R_(t)=0.94 min, m/z=264.3 (M−H),Method LCMS_2_MIN_FINAL_ANALYSIS. ¹H NMR (600 MHz, DMSO-d₆)* δ 12.62 (brs, 1 H), 7.44-7.09 (m, 5 H), 4.41 (d, J=8.1 Hz, 2 H) 1.44-1.24 (m, 9 H)3.89-3.67 (m, 2 H). *As a mixture with O(Boc)₂ (ca. 9%).

Intermediate E: tert-Butyl(2-(allyl(benzyl)amino)-2-oxoethyl)(benzyl)carbamate. A 1500 mL-4-neckreaction flask with mechanical stirrer, internal thermometer, condenserand nitrogen inlet was charged with intermediate D (31.6 g, 107 mmol)followed by EtOAc (500 ml). The reaction mixture was cooled in a icebath (4° C.) followed by addition of N-allylbenzylamine (16.44 g, 107mmol) and propylphosphonic anhydride (T3P, 136 g, 214 mmol, 50% in ethylacetate). To the mixture was added triethylamine (90 ml, 643 mmol),drop-wise over 5 min. The brown solution was stirred for 20 min at rtthen poured into a stirred mixture of ice water (500 ml). The phaseswere separated and the organic phase was washed successively with HCl(0.5 N, 500 mL), saturated NaHCO₃ (500 mL) and brine (500 mL). Theinitial aqueous layer was extracted with EtOAc (2×250 mL) and thecombined organic layers were dried over Na₂SO₄, filtered andconcentrated in vacuo at (45° C.), affording the title compound (43.94g) as a brown oil. LCMS: R_(t)=1.31, min m/z=395.5 (M+1), methodLCMS_2_MIN_FINAL_ANALYSIS. ¹H NMR (400 MHz, DMSO-d₆) δ 7.53-6.99 (m, 10H), 5.94-5.55 (m, 1 H), 5.24-4.97 (m, 2 H) 4.55-4.25 (m, 4 H), 4.16-3.68(m, 4 H), 1.42-1.28 (m, 9 H).

Intermediate F. N-ally-N-benzyl-2-(benzylamino)acetamide. A 750 ml4-neck reaction flask equipped with mechanical stirrer, internalthermometer, condenser and nitrogen inlet was charged with intermediateE (43.9 g, 108 mmol) in DCM (400 mL). To the solution was added TFA (83ml, 1.079 mol). After stirring o/n the yellow solution was slowly poured(rapid gas evolution) into a stirred mixture of saturated NaHCO₃solution (aq, 1.5 L) and ice (1 kg). After 10 min of stirring the phaseswere separated and the organic phase was washed with saturated NaHCO₃(aq, 0.5 L) then brine (0.5 L). The aqueous layer was extracted with DCM(0.5 L) and the combined organic layers were dried over Na₂SO₄, filteredand concentrated in vacuo (45° C.) to afford the title compound (31.30g) as a brown oil. LCMS: R_(t)=0.71 min m/z=295.3 (M+1), methodLCMS_2_MIN_FINAL_ANALYSIS. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.49-7.04 (m,10 H), 5.90-5.55 (m, 1 H), 5.19-4.92 (m, 2 H), 4.64-4.37 (m, 2 H),3.98-3.76 (m, 2 H), 3.73-3.61 (m, 2 H), 3.44-3.32 (m, 2 H), 2.44-2.28(m, 1 H).

Intermediate G: rac ethyl(2S*,3aS*,6aS*)-1,5-dibenzyl-6-oxooctahydropyrrolo[3,4-b]pyrrole-2-carboxylate.To a nitrogen inertized 60 L Buechi reactor CR60 equipped with a Huberthermostat 390W, Flexy ALR with automated temperature, dosage controland nitrogen inlet were added a solution of intermediate F (1.460 kg,4.81 mol) in toluene (20 L), magnesium sulfate (2.32 kg, 19.24 mol) andtriethylamine (0.872 L, 6.25 mol). The pale yellow suspension was heatedto reflux within 1 hour. To the refluxing mixture was added ethylglyoxylate (50% in toluene, 1.179 kg, 5.77 mol) over 15 h via a dosagepump. After stirring for an additional 6 h at reflux, the yellowsuspension was cooled to 15° C. (internal temp), whereupon water (20 L)was added (exothermic). After stirring for 15 min, the mixture wastransferred to a 80 L-separation vessel and the phases were separated.The organic layer was extracted successively with water (15 L) thenbrine (15 L). The aqueous layer was washed with TBME (2×10 L). Thesecond TBME wash was filtered through celite (contained insolublematerial), eluting with TBME. The combined organic phases were partiallyconcentrated in vacuo (45° C.) to a volume of 6 L, dried over Na₂SO₄,filtered and concentrated in vacuo (50° C.). This material was furtherdried overnight (50° C., 10 mbar) to afford the title compound (1.970kg) as a brown oil that was a 5.2:1 mixture of diastereomers. LCMS:R_(t)=1.21 min (67.1% a) m/z=379.3 (M+1); (12.9% a) at R_(t)=1.16 minm/z=379.3 (M+1), method LCMS_2_MIN_FINAL_ANALYSIS.

Intermediate H: rac(2S*,3aS*,6aS*)-1,5-dibenzyl-2-(hydroxymethyl)-hexahydropyrrolo-[3,4-b]pyrrol-6(1H)-one.To a solution of intermediate G (1.967 kg, 5.847 mol) in THF (20 L) at0° C. within a nitrogen inertized 30 L Buchi reactor CR30 equipped witha Huber thermostat 1015W, Flexy ALR, automated temperature control andnitrogen inlet was added lithium borohydride (0.238 kg, 10.39 mol) inportions over 10 min (slight exotherm). After 5 days at rt additionallithium borohydride (0.025 kg, 1.143 mol) was introduced. After anadditional 5 days at rt lithium borohydride (0.017 kg, 0.780 mol) wasadded. After 6 more days the mixture was cooled to −10° C., whereuponHCl (2 N, 8 L) was added dropwise via dosage pump over 2 h resulting ina pH=3 (caution: very strong gas and foam formation!). After vigorousstirring, a yellow suspension was formed that was stirred for 30 min at0° C. Saturated NaHCO₃ (aq, 10 L) was added and the mixture wastransferred to a 80 L-separation vessel and extracted with TBME (20 L)after addition of water (8 L), which aided the phase separation. Theorganic phase was washed with brine (2×10 L) and the aqueous layer wasextracted with TBME (2×7 L). The combined organic layers wereconcentrated in vacuo (45° C.) to a volume of 8 L, then dried overNa₂SO₄, filtered and concentrated in vacuo (50° C.). The residue wasdissolved in toluene (3 L), concentrated in vacuo and dried for 3 h (50°C., 10 mbar) to afford the title compound (1.630 kg) as a yellow-brownoil, which was a 10.5:1 mixture of diastereomers. LCMS: R_(t)=0.77 min*m/z=337.3 (M+1), method LCMS_2_MIN_FINAL_ANALYSIS. *Major diastereomer.

Intermediate I: rac(3R*,4aS*,7aS*)-1,6-dibenzyl-3-hydroxyoctahydro-7H-pyrrolo[3,4-b]pyridin-7-one.To a suspension of intermediate H (1.627 kg, 4.836 mol), molecularsieves (4 Å, 2.5 kg) and THF (23 L) in a nitrogen inertized 30 L triplejacketed Amsi Glas reactor equipped with automated temperature control,Unistat 390W, reflux condenser and nitrogen inlet at −5° C. was addedTFAA (0.820 L, 5.81 mol), dropwise over 35 min. After stirring for 15min at 0° C., triethylamine (3.37 L, 24.18 mol) was added over 10 min,whereupon it was heated to reflux (internal temperature 68° C.) for 6days, reaching an equilibrium ratio of product to starting material of9:1. The mixture was transferred into a 80 L separation vesselcontaining ice-cold NaOH (1 M, 24 L) and stirred for 15 min. To thebrown suspension was added celite (3 kg), where it was stirred for 15min then filtered over a pad of celite, washing with TBME. The filtratewas extracted with TBME (20 L). The organic phase was washed withsaturated NaHCO₃ (aq, 10 L) then brine (1×15 L). The aqueous layers wereextracted with TBME (2×7 L) and the combined organic layers wereconcentrated in vacuo (45° C.) to a volume of 8 L and dried over Na₂SO₄(2 kg). The suspension was filtered over silica gel (1 kg, 40-63 μm),washing with EtOAc (4×2 L). The eluent was concentrated in vacuo (45°C.) and dried for 3 h (50° C., 15 mbar) to afford the title compound(1.366 kg) as a dark brown oil. LCMS: R_(t)=0.84 min, m/z=337.3 (M+1),method LCMS_2_MIN_FINAL_ANALYSIS. ¹H NMR (600 MHz, DMSO-d₆) δ 7.40-7.15(m, 10 H), 4.69-4.57 (m, 1 H), 4.65-4.56 (m, 1 H), 4.43 (d, J=13.8 Hz, 1H), 4.29-4.21 (m, 1 H), 3.62-3.49 (m, 1 H), 3.46-3.39 (m, 1 H), 3.18 (d,J=8.3 Hz, 2 H), 2.86 (d, J=5.7 Hz, 1 H), 2.70 (dd, J=10.7, 2.7 Hz, 1 H),2.66-2.56 (m, 1 H), 1.83-1.67 (m, 2 H), 1.39-1.29 (m, 1 H).

Intermediate J:(3R,4aS,7aS)-1,6-dibenzyl-7-oxooctahydro-1H-pyrrolo[3,4-b]-pyridin-3-ylacetate. A suspension of intermediate I (1.364 kg, 3.04 mol), vinylacetate (4.20 L, 45.6 mol), Lipase QLM (Alcaligenes sp form MeitoSangyo, activity: 101400 U/g, 25 g, 3.04 mol) and TBME (21 L) in anitrogen inertized 30 L triple jacketed reactor with automatedtemperature control, Unistat 390W, condenser and nitrogen inlet wasstirred at 30° C. (internal temp) for 6 days. The mixture was cooled to20° C. and filtered over hyflo (500 g). The filtrate was concentrated invacuo (35° C.) to a volume of 3 L, whereupon toluene (1 L) was addedthen further concentrated in vacuo (35° C. then at 50° C.). The crudeproduct was dissolved in TBME:heptane (2:1, 3 L) and purified in severalportions via silica gel chromatography (heptane-EtOAc-methanol),affording the title compound (626 g) as a brown oil. LCMS: R_(t)=1.16min, m/z=379.3 (M+1), Method LCMS_2_MIN_FINAL_ANALYSIS. HPLC:R_(t)=33.45 min, 98.2% ee (minor enantiomer: R_(t)=23.92 min) methodHPLC_ CHIRAL. ¹H NMR (600 MHz, DMSO-d₆) δ ppm 7.41-7.22 (m, 10H),4.74-4.65 (m, 1H) 4.56-4.51 (m, 1H), 4.36-4.26 (m, 2H), 3.75 (d, J=14.3Hz, 1H) 3.30-3.21 (m, 2H) 3.00 (dd, J=9.5, 5.9 Hz, 1H), 2.75-2.68 (m,1H), 2.62 (sxt, J=6.2 Hz, 1H), 2.23 (dd, J=11.6, 7.2 Hz, 1H), 1.97 (s,3H), 1.66 (t, J=6.05 Hz, 2H).

Intermediate K:(3R,4aS,7aS)-1,6-dibenzyl-3-hydroxyoctahydro-7H-pyrrolo[3,4-b]-pyridin-7-one.A mixture of intermediate J (616 g, 1221 mmol), THF (4 L) and NaOH (2 N,3.97 L, 7.94 mol) in a 20 L round bottom flask of a Buchi Rotavapor wasvigorously stirred for 18 h at 25° C. and 6 h at 40° C., whereupon itwas cooled to 25° C. followed by addition of MeOH (2 L) and it wasstirred o/n. The mixture was extracted with TBME (6 L) and the organicphase was washed with brine (4 L). The aqueous layer was extracted withTBME (3×3 L) and the combined organic phases were concentrated in vacuo(45° C.) to a volume of 5 L, then dried over anhydrous sodium sulfate (1kg), filtered and concentrated in vacuo (45° C.). The residue wasdissolved in toluene (3 L) and reconcentrated then dried for 2 h (60°C., 20 mbar), affording the title compound (555 g) as a brown oil. LCMS:R_(t)=0.84 min, m/z=337.3 (M+1), Method LCMS_2_MIN_FINAL_ANALYSIS. ¹HNMR (600 MHz, DMSO-d₆) δ 7.58-7.04 (m, 10 H), 4.66-4.5 (m, 2 H), 4.43(d, J=13.9 Hz, 1 H), 4.25 (d, J=15.2 Hz, 1 H), 3.54 (tq, J=9.3, 4.5 Hz,1 H), 3.47-3.39 (m, 1 H), 3.18 (d, J=8.3 Hz, 2 H), 2.85 (d, J=5.9 Hz, 1H), 2.70 (dd, J=11.0, 2.8 Hz, 1 H), 2.64-2.57 (m, 1 H), 1.78-1.67 (m, 2H), 1.27-1.39 (m, 1 H).

Intermediate L:(3R,4aS,7aS)-1-benzyl-3-hydroxyoctahydro-7H-pyrrolo[3,4-b]-pyridin-7-one.To a 30 L Buchi reactor CR30 equipped with Huber thermostat 1015, FlexyALR with automated temperature control, argon and ammonia inlet wasinertized with argon, precooled to −80° C., filled with liquid ammonia(anhydrous, 10.0 kg, 587 mol), with the outlet attached to a gasscrubber filled with sulfuric acid (30%, 100 L), was added a solution ofintermediate K (543 g, 1.614 mol) in THF (1.5 L) followed by ethanol(anhydrous, 236 mL, 4.04 mol). To the resulting solution was addedlithium (granular, 44.8 g, 6.46 mol), portionwise over 15 min (tempraised from −72° C. to −63° C.). To the gray mixture, after 1 h, wasadded lithium (22.4 g, 3.23 mol) and ethanol (anhydrous, 94 mL, 1.616mol) while maintaining stirring at −60° C. After 1 h, additional lithium(11.2 g, 1.615 mol) and ethanol (anhydrous, 47 mL, 0.808 mol) wereadded. After 45 min more lithium (11.2 g, 1.615 mol) was added. After 15h ethanol (anhydrous, 94 mL, 1.616 mol) was added to the deep bluemixture. Stirring was continued until <5% starting material remained andit was quenched by addition of ammonium chloride (2.0 kg, 37.4 mol),portionwise over 10 min. The reaction mixture was stirred for 17 h at−28° C. and ˜2 h at 2° C., resulting in complete evaporation of theammonia. To the mixture was added water (15 L) and TBME (8 L) followedby HCl (32%) until pH=9-10 was obtained. The phases were separated andthe organic layer was washed with brine (5 L). The aqueous layer wasextracted with DCM (3×2 L) and the combined organic layers wereconcentrated in vacuo at 45° C. to a volume of 3 L then dried overNa₂SO₄ (1 kg), filtered and concentrated in vacuo (45° C. then 2 h at65° C., 20 mbar), affording the title compound (373 g) as a brown oil.LCMS: R_(t)=0.75, m/z=247.2 (M+H), Method LCMS_2_MIN_POLAR. ¹H NMR (600MHz, DMSO-d₆) δ 7.78 (s, 1 H), 7.34-7.24 (m, 4 H), 7.27-7.16 (m, 1 H),4.65-4.60 (m, 1 H), 4.32 (d, J=13.9 Hz, 1 H), 3.64-3.54 (m, 1 H), 3.44(d, J=13.9 Hz, 1 H), 3.13 (d, J=8.1 Hz, 2 H), 2.69 (dd, J=10.82, 2.75Hz, 1 H), 2.66-2.62 (m, 1 H), 2.60-2.54 (m, 1 H), 1.80-1.69 (m, 2 H),1.41-1.30 (m, 1 H).

Intermediate M: tert-butyl(3R,4aS,7aS)-3-hydroxy-7-oxooctahydro-1H-pyrrolo-[3,4-b]pyridine-1-carboxylate.To a solution of intermediate L (372.0 g, 1.51 mol) and Boc-anhydride(346 g, 1.59 mol) in THF (4.0 L) was added Pd—C 10% (15 g). The mixturewas agitated in a shaking duck apparatus at 22-25° C. and 0.1 bar H₂pressure for 89 h. After 57% hydrogen absorption another portion of Pd—C10% (15 g) was added. The mixture was filtered over celite, washed withTHF and concentrated in vacuo to obtain crude product (545 g) as a palebrown solid. The residue was suspended in EtOAc (1 L) and stirred for 1hour at 75° C. To the suspension was added heptane (1.5 L), slowly at75° C. After stirring for 2 h at rt, the product was collected byfiltration, the solid was washed with heptane then dried in vacuo (45°C.), affording to title compound (278.5 g) as white crystals. LCMS:R_(t)=0.86 min, m/z=257.3 (M+1), Method LCMS2_MIN_POLAR. ¹H NMR (600MHz, DMSO-d₆) δ 7.83-7.65 (m, 1 H), 4.80-4.49 (m, 2 H), 3.93-3.67 (m, 2H), 3.43-3.37 (m, 1 H), 2.72 (br d, J=9.5 Hz, 1 H), 2.60 (brd, J=12.5Hz, 1 H), 2.48-2.39 (m, 1 H), 1.82-1.70 (m, 1 H), 1.40 (brd, J=6.8 Hz, 9H) 1.36-1.27 (m, 1 H).

Intermediate N: tert-butyl(3S,4aS,7aS)-3-hydroxy-7-oxooctahydro-1H-pyrrolo-[3,4-b]pyridine-1-carboxylate.To a solution of intermediate M (270 g, 948 mmol) in THF (13 L)contained in a nitrogen inertized 20 L triple jacketed reactor (AmsiGlas) with automated temperature control, Unistat 390W, condenser andnitrogen inlet at −5° C. (internal temp) was added 4-nitrobenzoic acid(323 g, 1.90 mol) and triphenylphosphine (524 g, 1.90 mol). To theresulting solution was added a solution of DIAD (359 ml, 1.85 mol) inTHF (1.3 L), drop-wise over 30 min while maintaining the internal tempat −4 to −10° C. The mixture was allowed to warm to rt and stirred o/nthen concentrated in vacuo (45° C.) to provide crude material (1.64 kg,wet) as a brown oil. To a solution of oil residue in MeOH (15 L) wasadded K₂CO₃ (393 g, 2.844 mol). After 1 h of stirring the suspension wasconcentrated in vacuo (40° C.), providing an orange solid to which DCM(6 L) was added. After 30 min, the suspension was filtered, washing withDCM and the filtrate was concentrated in vacuo (45° C.). The residue wassuspended in DCM:MeOH (97:3, 4 L) and stirred for 30 min rt. Thesuspension was filtered, washing with DCM and the filtrate wasconcentrated in vacuo (45° C.) to a volume of 3 L and purified in twoportions by silica gel chromatography, affording product (189 g) assolid. To this material dissolved in DCM:MeOH (95:5, 5 L) at 45° C. wasadded heptane (5 L), slowly. The solution was partially concentrated(45° C.), removing some DCM, causing the product to crystallized after15 min. After 1 h at rt, the solid was collected via filtration, washedwith heptane and dried in vacuo (45° C.) until constant weight wasobtained, affording the title compound (167.7 g) as crystals. LCMS:R_(t)=0.95 min, m/z=257.3 (M+1), Method LCMS_2_MIN_POLAR. ¹H NMR (600MHz, DMSO-d₆)* δ 7.80 (d, J=20.2 Hz, 1H), 4.97 (t, J=4.8 Hz, 1H), 4.57(dd, J=92.2, 7.0 Hz, 1H), 3.89 (ddd, J=18.8, 9.3, 6.5 Hz, 1H), 3.33-3.23(m, 1H), 2.75 (ddd, J=9.7, 4.6, 2.0 Hz, 1H), 2.43 (ddt, J=16.9, 11.8,6.1 Hz, 1H), 2.08 (ddd, J=86.6, 12.5, 10.7 Hz, 1H), 1.94 (dd, J=12.2,5.5 Hz, 1H), 1.39 (d, J=22.6 Hz, 9H), 1.04 (dq, J=15.2, 12.0 Hz, 1H).*Reported as observed rotamers.

EXAMPLE 1 Sodium(4R,5aS,8aS)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate.

Step 1: Methyl(2S,3S,5R)-6-(benzyloxy)-3-(((tert-butoxycarbonyl)amino)-methyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate.Intermediate C (0.82 g, 2.84 mmol), Boc-Gly-OH (1.00 g, 5.69 mmol) andIr[df(CF₃)ppy₂(dtbpy)]PF₆ (32 mg, 0.028 mmol) were dissolved in DMF (20mL). To the solution was added finely ground potassium phosphate dibasic(0.59 g, 3.41 mmol) and the resulting suspension was irradiated underargon (balloon) in a 500 mL dropping funnel (closed with a round bottomflask at the bottom and a septum at the top) for 7 days with a 8W UVAfluorescence tube. The flask was placed horizontally on the top of thelamp (air cooled) to ensure maximum irradiation. After 4 daysIr[df(CF₃)ppy₂(dtbpy)]PF₆ (32 mg, 0.028 mmol) was added.

To the mixture were added water (100 mL) then saturated NaHCO₃ (aq, 100mL) and it was extracted with TBME (4×80 mL). The combined organicphases were washed sequentially with saturated NaHCO₃ (aq, 50 mL), water(50 mL) then brine (50 mL). The organic layer was dried over Na₂SO₄,filtered and concentrated in vacuo. The crude residue was purified viasilica gel chromatography (EtOAc-heptane, 15-100%) to afford the titlecompound (108 mg, 9%) as an oil. LCMS: R_(t)=1.04 min, Method 2m_acidic.

Step 2:(4R,5aS,8aS)-3-(benzyloxy)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione.To a solution of methyl(2S,3S,5R)-6-(benzyloxy)-3-(((tert-butoxycarbonyl)amino)-methyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate(108 mg, 0.26 mmol) in DCM (3 mL) at rt was added TFA (1.0 mL, 13 mmol),drop-wise. It was allowed to stir at rt for 3 h, whereupon it wasconcentrated in vacuo. The crude residue was dissolved in DCM (3 mL),cooled to 0° C., and triethylamine (0.31 mL, 2.3 mmol) was added. After1 h more triethylamine (0.11 mL, 0.75 mmol) and DCM (5 mL) were added.The ice bath was removed and the reaction mixture was stirred at rtovernight (o/n), whereupon it was washed with citric acid (10 mL, ca 20%aq). The aqueous phase was extracted with DCM (3×8 mL) and the combinedorganic phases were washed with water (5 mL), brine (2×10 mL), driedover Na₂SO₄, filtered and concentrated in vacuo. The crude residue waspurified via silica gel chromatography (DCM-MeOH, 2-7%) to afford thetitle compound (47 mg, 61%) as a beige solid. LCMS: R_(t)=0.61 min,m/z=288 (M+1), Method 2m_acidic.

Step 3:(4R,5aS,8aS)-3-hydroxyhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione.A slurry of(4R,5aS,8aS)-3-(benzyloxy)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione(80 mg, 0.278 mmol) and Pd—C (10% Degussa type 101, 50% water, 34 mg) inMeOH (1.8 mL) was evacuated and backfilled with H₂ (3×). After 2.5 h itwas filtered through a plug of celite, washed with MeOH and concentratedin vacuo, affording the title compound (40 mg, 74%). LCMS: R_(t)=0.13min, m/z=198.1 (M+1) Method 2m_acidic.

Step 4: Sodium(4R,5aS,8aS)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. To a slurry of crude(4R,5aS,8aS)-3-hydroxyhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione(40.7 mg, 0.206 mmol) in pyridine (2 mL) at 0° C. was added SO₃.Pycomplex (335 mg, 2.064 mmol). After 19 h of vigorous stirring the slurrywas filtered and concentrated in vacuo. The crude residue was dissolvedin THF:water (1:1, 6 mL) and Amberlite 200 Na-exchange resin (1.5 g) wasadded. The suspension was stirred for 2 h, whereupon it was filtered,partially concentrated in vacuo, frozen and lyophilized. The resultingsolid was subjected to silica gel chromatography (water-acetonitrile,2-5%), affording the title compound (12.3 mg, 16%, over 2-steps) as anamorphous solid. LCMS: R_(t)=0.25 min, m/z=278.0 (M+1) MethodT3_3m_polar; ¹H NMR (500 MHz, D₂O) δ 4.24-4.18 (m, 2H), 3.52 (dd,J=10.7, 6.2 Hz, 1H), 3.33 (d, J=12.3 Hz, 1H), 3.10 (d, J=10.7 Hz, 1H),2.95 (d, J=12.3 Hz, 1H), 2.81 (p, J=8.5 Hz, 1H), 2.54-2.46 (m, 1H), 1.65(dd, J=14.7, 9.2 Hz, 1H).

EXAMPLE 1 Alternate Procedure. Sodium(4R,5aS,8aS)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate.

Step 1: (2S,3S,5R)-methyl6-(benzyloxy)-3-(((tert-butoxycarbonyl)amino)methyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate.A stirred mixture of Intermediate C (3 g, 10.41 mmol),2-((tert-butoxycarbonyl)amino)acetic acid (2.55 g, 14.57 mmol),Ir[df(CF₃)ppy]₂(dtbpy)PF₆ (0.117 g, 0.104 mmol), and potassium phosphatedibasic (2.72 g, 15.61 mmol) in DMF (30.6 mL) was degassed via N₂ spargefor 15 min and irradiated with a Kessil H150-Blue LED (fan cooling),under N₂ for 92 h. 2-((tert-butoxycarbonyl)amino)acetic acid (2.55 g,14.57 mmol), potassium phosphate dibasic (2.72 g, 15.61 mmol), andIr[df(CF₃)ppy]₂(dtbpy)PF₆ (0.117 g, 0.104 mmol) were added and themixture was irradiated with a Kessil H150-Blue LED (fan cooling), underN₂ for an additional 20 h. The mixture was diluted with saturated NaHCO₃(aq) and extracted with EtOAc (3×). The combined organic layers werewashed with water, brine, dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The crude material was purified by silica gelchromatography (EtOAc-Heptanes, 0-100%) to afford the title compound(352 mg, 8%) as a yellow foam. LC/MS: R_(t)=0.87 min; m/z=420.2 (M+1)Method 2m_acidic; ¹H NMR (500 MHz, DMSO-d₆) δ 7.46-7.42 (m, 2H),7.42-7.34 (m, 3H), 6.84 (br s, 1H), 4.93 (d, J=4.3 Hz, 2H), 3.88 (d,J=6.7 Hz, 1H), 3.76 (br s, 1H), 3.66-3.65 (m, 3H), 3.21 (d, J=12.0 Hz,1H), 3.12-3.03 (m, 1H), 2.86-2.78 (m, 2H), 2.14 (br s, 1H), 2.00-1.93(m, 1H), 1.51 (t, J=12.3 Hz, 1H), 1.34 (s, 9H)

Step 2:(4R,5aS,8aS)-3-(benzyloxy)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione.To a solution of (2S,3S,5R)-methyl6-(benzyloxy)-3-(((tert-butoxycarbonyl)amino)methyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate(352 mg, 0.839 mmol) in DCM (4.19 mL) was added TFA (1.61 mL, 20.98mmol), drop-wise. After 90 min it was concentrated in vacuo, dissolvedin DCM and reconcentrated (3×). To the residue, dissolved in DCM (5 mL)at 0° C. was added TEA (1.17 mL, 8.39 mmol), after which the coolingbath was removed. After 20 h at rt, the mixture was diluted withsaturated NaHCO₃ (aq) and extracted with EtOAc (3×). The combinedorganic layers were washed with water, brine, dried over Na₂SO₄,filtered and concentrated in vacuo. The crude material was purified bysilica gel chromatography (MeOH-DCM, 0-20%), affording the titlecompound (158 mg, 66%, 2-steps) as a clear film. LC/MS: R_(t)=0.65 min;m/z=288.0 (M+1) Method 2m_acidic; ¹H NMR (400 MHz, DMSO-d₆) δ 7.99 (s,1H), 7.48-7.33 (m, 5H), 4.99-4.88 (m, 2H), 3.81 (d, J=7.8 Hz, 1H), 3.59(br s, 1H), 3.32-3.22 (m, 1H), 2.89 (br d, J=11.9 Hz, 1H), 2.75 (d,J=9.8 Hz, 1H), 2.63 (d, J=11.9 Hz, 1H), 2.26-2.17 (m, 1H), 1.38 (ddd,J=14.3, 9.2, 1.9 Hz, 1H)

Step 3:(4R,5aS,8aS)-3-hydroxyhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione.A slurry of(4R,5aS,8aS)-3-(benzyloxy)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione(158 mg, 0.550 mmol) and Pd—C (10% Degussa type 101, 50% water, 117 mg,0.055 mmol) in MeOH:DCM (3:1, 3.67 mL) was evacuated and backfilled withH₂. After 2 h, the mixture was filtered through celite and concentratedin vacuo (bath temp <30° C.) to afford the title compound (102 mg, 94%)as an off-white solid. LC/MS: R_(t)=0.12 min; m/z=198.0 (M+1) Method2m_acidic.

Step 4: Tetrabutylammonium(4R,5aS,8aS)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. To a solution of crude(4R,5aS,8aS)-3-hydroxyhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione(102 mg, 0.517 mmol) in Pyridine (5.17 mL) was added SO₃.Py (412 mg,2.59 mmol). After 19 h of vigorous stirring, the mixture was filteredand concentrated in vacuo (bath temp <30° C.). The resulting materialwas dissolved in NaH₂PO₄ (1 M, 10 mL), whereupon tetrabutylammoniumhydrogen sulfate (263 mg, 0.776 mmol) was added. After 30 min ofstirring it was extracted with IPA:CHCl₃ (1:4, 3×). The combined organiclayers were dried over Na₂SO₄, filtered and concentrated in vacuo (bathtemp <30° C.). The crude residue was purified by silica gelchromatography (MeOH-DCM, 0-30%) to afford the title compound (180 mg,67%) as a white foam. LC/MS: R_(t)=0.13 min; m/z=278 (M+1) Method2m_acidic; ¹H NMR (500 MHz, DMSO-d₆) δ 7.97 (s, 1H), 3.98 (br s, 1H),3.81 (d, J=7.8 Hz, 1H), 3.28 (dd, J=6.1, 9.9 Hz, 1H), 3.19-3.13 (m, 8H),2.98 (br d, J=12.1 Hz, 1H), 2.78 (d, J=9.9 Hz, 1H), 2.65 (d, J=12.1 Hz,1H), 2.49-2.44 (m, 1H), 2.28-2.19 (m, 1H), 1.63-1.51 (m, 8H), 1.40 (brdd, J=9.3, 12.7 Hz, 1H), 1.31 (sxt, J=7.4 Hz, 8H), 0.93 (t, J=7.3 Hz,12H)

Step 5: Sodium(4R,5aS,8aS)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. DOWEX 50Wx8 hydrogen form 200-400 mesh was conditioned bystirring with NaOH (2 N) for 3 h. The resin was loaded onto a column andwashed with water until the pH was ˜6. It was then washed with 1:1water/acetone. tetrabutylammonium(4R,5aS,8aS)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate (180 mg, 0.347 mmol) was dissolved in 1:1 acetone/water andeluted through the resin with 1:1 acetone/water. The sample waspartially concentrated in vacuo (bath temp <30° C.), and lyophilized toafford the title compound (75 mg, 68%) as a white solid. LC/MS:R_(t)=0.25 min; m/z=278.0 (M+1) Method T3_3m_polar; ¹H NMR (500 MHz,D₂O) δ 4.24-4.18 (m, 2H), 3.51 (dd, J=10.7, 6.2 Hz, 1H), 3.33 (brd,J=12.3 Hz, 1H), 3.10 (d, J=10.7 Hz, 1H), 2.95 (d, J=12.3 Hz, 1H), 2.81(p, J=8.1 Hz, 1H), 2.54-2.46 (m, 1H), 1.65 (dd, J=14.7, 9.2 Hz, 1H).

Alternate Step 5 Procedure:(4R,5aS,8aS)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylhydrogen sulfate

To a solution of tetrabutylammonium(4R,5aS,8aS)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate (6.9 g, 13.30 mmol) in isobutanol (20.8 mL) and water (1.35 mL)at 40° C. was added a solution of sodium 2-ethylhexanoate (4.56 g, 26.6mmol) in isobutanol (20.8 mL) and water (1.35 mL) via syringe pump at 8mL/h. The mixture was stirred for 1 h at 40° C. then cooled to rt andstirred overnight, whereupon it was filtered with a Buchner funnel usingWhatman qualitative filter paper. The filter cake was washed withisobutanol (3×) and then ice-cold acetone (3×). Vacuum was applied tothe funnel with N₂ stream over the filter cake for 3 h followed bylyophilization for 3 days, which afforded the title compound (3.05 g,73%) as a crystalline white solid. LC/MS: R_(t)=0.25 min; m/z=278.0(M+1) Method T3_3m_polar.

¹H NMR (500 MHz, D₂O) δ=4.20-4.12 (m, 2H), 3.48 (dd, J=6.2, 10.7 Hz,1H), 3.33-3.25 (m, 1H), 3.05 (d, J=10.7 Hz, 1H), 2.90 (d, J=12.2 Hz,1H), 2.82-2.71 (m, 1H), 2.46 (tdd, J=2.8, 8.7, 14.7 Hz, 1H), 1.66-1.56(m, 1H)

The X-ray powder diffraction spectrum for the sodium salt is shown inFIG. 1.

Instrument: X-Ray Diffractometer (Bruker, model D8)

Source—Cu k α

Step width 0.02°

Voltage 40 kV

Current 40 mA

Time per step 120 seconds

Scan Range 3 to 39°

Peak 2theta 1  8.31 ± 0.2) 2 11.66 ± 0.2) 3 14.45 ± 0.2) 4 16.63 ± 0.2)5 17.64 (± 0.2)   6 18.12 ± 0.2) 7 18.64 ± 0.2) 8 19.51 ± 0.2) 9 21.68 ±0.2) 10 23.54 ± 0.2) 11 24.32 ± 0.2) 12 25.06 (± 0.2)   13 27.37 ± 0.2)14 27.86 ± 0.2) 15 28.72 ± 0.2) 16 31.33 ± 0.2) 17 32.60 ± 0.2) 18 33.58± 0.2) 19 34.43 ± 0.2) 20 35.40 ± 0.2) 21 38.17 (± 0.2)  

EXAMPLE 2 Sodium(4R,5aS,8aS)-7-methyl-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate.

Step 1: (2S,3S,5R)-methyl6-(benzyloxy)-3-(((tert-butoxycarbonyl)(methyl)amino)methyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate.A stirred mixture of Intermediate C (3 g, 10.41 mmol),2-((tert-butoxycarbonyl)(methyl)amino)acetic acid (2.56 g, 13.53 mmol),Ir[df(CF₃)ppy]₂(dtbpy)PF₆ (0.117 g, 0.104 mmol), and potassium phosphatedibasic (2.175 g, 12.49 mmol) in DMF (30 mL) was degassed by bubbling N₂through the suspension for 15 min and then left under an N₂ line andirradiated with a Kessil H150-Blue LED (fan cooling) for 48 h. Thereaction was diluted with saturated NaHCO₃ and extracted with EtOAc(3×). The combined organic layers were washed with water, brine, driedover Na₂SO₄, filtered and concentrated in vacuo. The crude material waspurified by silica gel chromatography (EtOAc-Heptanes, 0-100%) to affordan orange foam (233 mg). This material was repurified by silica gelchromatography (EtOAc-Heptanes, 0-70%) affording the title compound (140mg, 3%) as a yellow solid. LC/MS: R_(t)=0.92 min; m/z=434.1 (M+1) Method2m_acidic; ¹H NMR (500 MHz, DMSO-d₆) δ 7.46-7.42 (m, 2H), 7.41-7.33 (m,3H), 4.98-4.91 (m, 2H), 3.88-3.81 (m, 1H), 3.78 (br s, 1H), 3.68 (br s,3H), 3.26-3.03 (m, 2H), 2.84 (br d, J=11.6 Hz, 1H), 2.67 (s, 3H),1.97-1.88 (m, 1H), 1.62 (br t, J=12.6 Hz, 1H), 1.34 (br s, 9H)

Step 2: (2S,3S,5R)-methyl6-(benzyloxy)-3-((methylamino)methyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate.To a stirred solution of (2S,3S,5R)-methyl6-(benzyloxy)-3-(((tert-butoxycarbonyl)(methyl)amino)methyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate(140 mg, 0.323 mmol) in DCM (1.6 mL), TFA (0.622 mL, 8.07 mmol) wasadded drop wise at rt under N₂. The reaction was stirred at rt for 90min and then concentrated and coevaporated with DCM (3×). The residuewas dissolved in DCM (2 mL) and cooled to 0° C. TEA (0.450 mL, 3.23mmol) was added, the cooling bath removed and the reaction stirred at rtfor 90 min. The reaction was diluted with saturated NaHCO₃ and extractedwith EtOAc (3×). The combined organic layers were washed with water,brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The crudematerial was purified by silica gel chromatography (MeOH-DCM, 0-20%) toafford the title compound (86 mg, 88%) as a clear film. LC/MS:R_(t)=0.46 min; m/z=301.9 (M+1) Method 2m_acidic; ¹H NMR (400 MHz,DMSO-d₆) δ 7.50-7.32 (m, 5H), 5.03-4.86 (m, 2H), 3.86 (d, J=7.9 Hz, 1H),3.60 (br s, 1H), 3.39-3.34 (m, 1H), 2.93-2.83 (m, 2H), 2.76 (s, 3H),2.57 (d, J=11.9 Hz, 1H), 2.49-2.42 (m, 1H), 2.32-2.18 (m, 1H), 1.38(ddd, J=14.3, 9.0, 1.9 Hz, 1H)

Step 3:(4R,5aS,8aS)-3-hydroxy-7-methylhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione.(2S,3S,5R)-methyl6-(benzyloxy)-3-((methylamino)methyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate(143 mg, 0.475 mmol) was dissolved in methanol (2.4 mL) and Pd—C (10%,Degussa type 101, 50% water, 101 mg, 0.047 mmol) was added. The mixturewas evacuated under vacuum and backfilled with H₂. After 1 h ofstirring, the mixture was filtered through celite and concentrated invacuo (bath temp <30° C.) to afford the title compound (63 mg, 63%) as awhite solid. LC/MS: R_(t)=0.11 min; m/z=211.9 (M+1) Method 2m_acidic.

Step 4: tetrabutylammonium(4R,5aS,8aS)-7-methyl-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate.(4R,5aS,8aS)-3-hydroxy-7-methylhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione(63 mg, 0.298 mmol) was dissolved in Pyridine (2.9 mL) and SO₃.pyridine(237 mg, 1.49 mmol) was added. The reaction was stirred at rt for 20 h.The reaction was filtered through a disposable plastic filter andconcentrated under reduced pressure (bath temp <30° C.). This materialwas dissolved in NaH₂PO₄ (1 M, 10 mL) and tetrabutylammonium hydrogensulfate (152 mg, 0.447 mmol) was added. After stirring for 30 min at rtit was extracted with EtOAc (4×). The combined organic layers werewashed with brine, dried over Na₂SO₄, filtered and concentrated invacuo. The aqueous was further extracted with 20% IPA in CHCl₃ (2×),dried over sodium sulfate and concentrated in vacuo (bath temp <30° C.).The combined organic material was purified by silica gel chromatography(MeOH-DCM, 0-30%) to afford the title compound (62 mg, 39%) as a clearfilm. LC/MS: R_(t)=0.13 min; m/z=291.9 (M+1) Method 2m_acidic; ¹H NMR(400 MHz, CDCl₃) δ=4.34 (br s, 1H), 4.04 (br d, J=7.3 Hz, 1H), 3.44 (dd,J=10.0, 5.6 Hz, 1H), 3.34 (brd, J=2.6 Hz, 1H), 3.28 (br dd, J=10.3 Hz,5.1 Hz, 8H), 2.97-2.91 (m, 4H), 2.80 (d, J=12.0 Hz, 1H), 2.75-2.61 (m,2H), 1.74-1.60 (m, 9H), 1.44 (sxt, J=7.4 Hz, 8H), 1.00 (t, J=7.3 Hz,12H).

Step 5: sodium(4R,5aS,8aS)-7-methyl-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. DOWEX 50Wx8 hydrogen form 200-400 mesh was conditioned bystirring with NaOH (2 N) for 3 h. The resin was loaded onto a glasscolumn and washed with water until the pH was ˜6. It was then washedwith water:acetone (1:1). Tetrabutylammonium(4R,5aS,8aS)-7-methyl-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate (62 mg, 0.12 mmol) was dissolved in 1:1 acetone:water and passeddown the column with acetone:water (1:1). The sample was partiallyconcentrated in vacuo (bath temp <30° C.) then lyophilized to afford thetitle compound (32 mg, 83%) as a white powder. LC/MS: R_(t)=0.48 min;m/z=291.8 (M+1) Method T3_3m_polar; ¹H NMR (500 MHz, D₂O) δ=4.20-4.15(m, 2H), 3.60 (dd, J=10.6, 6.4 Hz, 1H), 3.30 (dddd, J=12.3, 4.0, 2.7,1.3 Hz, 1H), 3.13 (d, J=10.4 Hz, 1H), 2.90 (s, 3H), 2.86 (d, J=12.1 Hz,1H), 2.77-2.69 (m, 1H), 2.49 (tdd, J=14.8, 8.8, 3.0 Hz, 1H), 1.59 (ddd,J=14.8, 8.9, 2.0 Hz, 1H).

EXAMPLE 3 Sodium(4R,5aS,8aS)-7-cyclopropyl-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate.

Step 1: (2S,3S,5R)-methyl6-(benzyloxy)-3-(((tert-butoxycarbonyl)(cyclopropyl)amino)methyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate.A stirred mixture of Intermediate C (4 g, 13.87 mmol),2-((tert-butoxycarbonyl)(cyclopropyl)amino)acetic acid (3.88 g, 18.04mmol), Ir[df(CF₃)ppy₂(dtbpy)]PF₆ (0.156 g, 0.139 mmol), and potassiumphosphate dibasic (2.90 g, 16.65 mmol) in DMF (40 mL) was degassed viaN₂ sparge for 15 min. The mixture was irradiated under N₂ with a KessilH150-Blue LED (fan cooling) for 42 h, whereupon it was was diluted withsaturated NaHCO₃, filtered and extracted with EtOAc (3×). The combinedorganic layers were washed with water, brine, dried over Na₂SO₄,filtered and concentrated in vacuo. The crude residue was purified bysilica gel chromatography (EtOAc-Heptanes, 0-100%). This material wasrepurified 2 more times by silica gel chromatography (EtOAc-Heptanes,0-100% then EtOAc-Heptanes, 0-70%) affording the title compound (190 mg,3% Yield) as a clear film. LC/MS: R_(t)=0.99 min; m/z=460.2 (M+1) Method2m_acidic; ¹H NMR (400 MHz, CDCl₃) δ=7.45-7.34 (m, 5H), 5.05 (d, J=11.5Hz, 1H), 4.89 (d, J=11.5 Hz, 1H), 4.09 (d, J=6.5 Hz, 1H), 3.74 (s, 3H),3.37-3.25 (m, 2H), 3.17-3.07 (m, 1H), 2.92-2.83 (m, 1H), 2.64-2.52 (m,1H), 2.44-2.35 (m, 1H), 2.09-1.98 (m, 1H), 1.68 (t, J=12.7 Hz, 1H),1.58-1.52 (m, 1H), 1.44-1.39 (m, 9H), 0.80-0.66 (m, 2H), 0.59-0.46 (m,2H)

Step 2:(4R,5aS,8aS)-3-(benzyloxy)-7-cyclopropylhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione.To a stirred solution of (2S,3S,5R)-methyl6-(benzyloxy)-3-(((tert-butoxycarbonyl)(cyclopropyl)amino)methyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate(190 mg, 0.413 mmol) dissolved in DCM (4.1 mL), TFA (0.796 mL, 10.34mmol) was added drop-wise at rt under N₂. The solution was stirred at rtfor 90 min and then concentrated in vacuo, diluted with DCM andreconcentrated (3×). The residue was dissolved in DCM (2 mL) and cooledto 0° C. TEA (0.576 mL, 4.13 mmol) was added and it was stirred for 18 hat rt. The solution was concentrated under reduced pressure and thecrude material was purified by silica gel chromatography (MeOH-DCM,0-20%), affording the title compound (103 mg, 76%) as a clear film.LC/MS: R_(t)=0.69 min; m/z=328.0 (M+1) Method 2m_acidic; ¹H NMR (500MHz, DMSO-d₆) δ=7.46-7.42 (m, 2H), 7.41-7.33 (m, 3H), 4.97-4.89 (m, 2H),3.87 (d, J=7.8 Hz, 1H), 3.59 (br s, 1H), 3.31-3.27 (m, 1H), 2.91-2.85(m, 1H), 2.76 (d, J=9.8 Hz, 1H), 2.66 (tt, J=7.5, 4.1 Hz, 1H), 2.56 (d,J=11.9 Hz, 1H), 2.42 (dd, J=8.5, 6.1 Hz, 1H), 2.19 (ddt, J=14.3, 8.5,3.0 Hz, 1H), 1.31 (ddd, J=14.3, 9.2, 1.9 Hz, 1H), 0.78-0.68 (m, 2H),0.66-0.55 (m, 2H).

Step 3:(4R,5aS,8aS)-7-cyclopropyl-3-hydroxyhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione.(4R,5aS,8aS)-3-(benzyloxy)-7-cyclopropylhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione(103 mg, 0.315 mmol) was dissolved in MeOH (3.2 mL) and Pd—C (10%Degussa type 101, 50% water, 67.0 mg, 0.031 mmol) was added. The mixturewas degassed in vacuo and backfilled with H₂. After stirring for 40 min,it was filtered through celite and concentrated in vacuo (bath temp <30°C.) to afford the title compound (75 mg, 100%) as a white solid. LC/MS:R_(t)=0.54 min; m/z=238.0 (M+1) Method 2m_acidic.

Step 4: Tetrabutylammonium(4R,5aS,8aS)-7-cyclopropyl-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. To a solution of(4R,5aS,8aS)-7-cyclopropyl-3-hydroxyhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione(75 mg, 0.316 mmol) in pyridine (3.16 mL) was added SO₃.pyridine (151mg, 0.948 mmol). After stirring for 20 h, the slurry was filtered andconcentrated in vacuo (bath temp <30° C.). The crude residue wasdissolved in saturated NaH₂PO₄ (10 mL) and washed with EtOAc. To theaqueous layer was added tetrabutylammonium hydrogen sulfate (161 mg,0.474 mmol). After stirring for 45 min it was extracted with DCM (4×),dried over Na₂SO₄, filtered and concentrated in vacuo (bath temp <30°C.). The crude residue was purified via silica gel chromatography(Acetone-DCM, 0-100%) to afford 121 mg of a clear film. LC/MS:R_(t)=0.14 min; m/z=318.0 (M+1) Method 2m_acidic; ¹H NMR (400 MHz,DMSO-d₆) δ=3.93 (br s, 1H), 3.87 (d, J=7.8 Hz, 1H), 3.34-3.29 (m, 1H),3.19-3.13 (m, 8H), 2.99-2.94 (m, 1H), 2.78 (d, J=9.7 Hz, 1H), 2.68 (tt,J=7.4, 4.2 Hz, 1H), 2.57 (d, J=11.9 Hz, 1H), 2.46-2.36 (m, 1H),2.26-2.17 (m, 1H), 1.61-1.52 (m, 8H), 1.35-1.26 (m, 9H), 0.93 (t, J=7.3Hz, 12H), 0.78-0.69 (m, 2H), 0.66-0.57 (m, 2H).

Step 5: Sodium(4R,5aS,8aS)-7-cyclopropyl-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. DOWEX 50Wx8 hydrogen form 200-400 mesh was conditioned bystirring with NaOH (2 N) for 3 h. The resin was loaded onto a glasscolumn and washed with water (until pH=6) followed by water:acetone(1:1). A solution of tetrabutylammonium(4R,5aS,8aS)-7-cyclopropyl-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate (121 mg, 0.217 mmol) in water:acetone (1:1) was loaded onto andpassed through the column, eluting with water:acetone (1:1). The samplewas concentrated in vacuo (bath temp <30° C.) and lyophilized, affordingthe title compound (51 mg, 66% Yield) as a white powder. LC/MS:R_(t)=0.36 min; m/z=318.0 (M+1) Method 2m_acidic; ¹H NMR (500 MHz, D₂O)δ 4.20-4.14 (m, 2H), 3.55 (dd, J=10.6, 6.1, Hz, 1H), 3.32-3.26 (m, 1H),3.09 (d, J=10.6 Hz, 1H), 2.83 (d, J=12.3 Hz, 1H), 2.73-2.65 (m, 2H),2.50-2.42 (m, 1H), 1.51 (dd, J=14.6, 9.1 Hz, 1H), 0.90-0.74 (m, 3H),0.71-0.63 (m, 1H).

EXAMPLE 4 Sodium(4R,5aS,8aS)-7-(2-hydroxyethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate.

Step 1: tert-butyl(3S,4aS,7aS)-6-allyl-3-hydroxy-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate.To a soln of tert-butyl(3S,4aS,7aS)-3-hydroxy-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate(1.50 g, 5.62 mmol) in DMF (56 mL) at 0° C. was added Potassiumtert-butoxide (1 M in THF, 5.6 ml, 5.6 mmol). After 5 min the cold bathwas removed and it was allowed to stir at rt for 30 min then cooled to0° C., whereupon allylbromide (490 μL, 5.66 mmol) was added drop-wise.The cold bath was removed after 5 min and after an additional 2 h at rt,it was concentrated in vacuo and purified directly via silica gelchromatography (ethylacetate-heptane, 0-100%) to afford the titlecompound (1.688 g, 81%) as a white solid. LC/MS: R_(t)=0.70 min;m/z=297.1 (M+1) Method 2m_acidic; ¹H NMR (500 MHz, DMSO-d₆)* δ=5.72(ddt, J=16.4, 11.1, 5.9 Hz, 1H), 5.23-5.15 (m, 2H), 4.96 (s, 1H), 4.77(d, J=7.1 Hz, 0.5H), 4.62 (d, J=7.1 Hz, 0.5H), 3.97-3.85 (m, 1.5H),3.85-3.75 (m, 1H), 3.71 (dd, J=15.3, 6.3 Hz, 0.5H), 3.49-3.42 (m, 1H),2.83-2.77 (m, 1H), 2.50-2.40 (m, 1H), 2.14 (t, J=11.6 Hz, 0.5H),2.03-1.91 (m, 1.5H), 1.42 (s, 4.5H), 1.38 (s, 4.5H) 0.96 (p, J=12.1 Hz,1H). *Reported as a mixture of rotamers.

Step 2: tert-butyl(3S,4aS,7aS)-3-hydroxy-6-(2-hydroxyethyl)-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate.A solution of tert-butyl(3S,4aS,7aS)-6-allyl-3-hydroxy-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate(2.72 g, 9.18 mmol) in DCM (92 ml) at −78° C. was sparged with O₃ for 30min. It was then purged by bubbling O₂ for an additional 20 min at −78°C. To the clear soln was added dimethylsulfide (6.74 ml, 92 mmol) and itwas warmed to rt and stirred for 30 min. It was cooled to 0° C. and MeOH(18 mL) was added followed by sodium borohydride (694 mg, 18.4 mmol)then allowed to slowly warm to rt. After 14 h at rt it was cooled to 0°C. and saturated NH₄Cl (aq, 10 mL) was added. After 20 min at rt it wasconcentrated in vacuo followed by addition of MeOH and reconcentrated.The residue was taken up in MeOH, filtered then reconcentrated. Tolenewas added and the slurry was sonicated then reconcentrated. LCMS:R_(t)=0.40 min; m/z=301.4 (M+1) Method 2m_acidic.

Step 3: tert-butyl(3S,4aS,7aS)-6-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-hydroxy-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate.A soln of tert-butyl(3S,4aS,7aS)-3-hydroxy-6-(2-hydroxyethyl)-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate(9.18 mmol) in pyridine (18 ml) was added TBS-Cl (1.384 g, 9.18 mmol).After stirring for 24 h at rt it was concentrated in vacuo and taken upin EtOAc and washed with water. The aqueous layer was extracted withEtOAc (2×) and the combined organic layers were washed with brine, driedover Na₂SO₄, filtered and concentrated in vacuo. The crude residue waspurified via silica gel chromatography, affording the title compound(2.433 g, 64% 3-steps) as a white solid. LCMS: R_(t)=0.89 min; m/z=415.4(M+1) Method 2m_acidic.

Step 4: tert-butyl(3R,4aS,7aS)-3-((N-(benzyloxy)-2-nitrophenyl)sulfonamido)-6-(2-((tert-butyldimethylsilyl)oxy)ethyl)-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate.To a solution of tert-butyl(3S,4aS,7aS)-6-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-hydroxy-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate(2.411 g, 5.82 mmol), N-(benzyloxy)-2-nitrobenzenesulfonamide (2.160 g,7.01 mmol) and triphenylphosphine (1.830 g, 6.98 mmol) in THF (65 ml) at−17° C. was added DIAD (1.40 ml, 6.98 mmol) as a soln in THF (10 mL),drop-wise. It was allowed to slowly warm to rt and stir for 18 h thenconcentrated in vacuo and purified directly via silica gelchromatography, affording the title compound (1.785 g, 44% yield) as awhite solid. LCMS: R_(t)=1.15 min; m/z=705.4 (M+1) Method 2m_acidic.

Step 4:N-(benzyloxy)-N-((3R,4aS,7aS)-6-(2-((tert-butyldimethylsilyl)oxy)ethyl)-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridin-3-yl)-2-nitrobenzenesulfonamide.To a flask charged with zinc(II) bromide (1.21 g, 5.37 mmol, dried at200° C. for 3 h, was added a solution of tert-butyl(3R,4aS,7aS)-3-((N-(benzyloxy)-2-nitrophenyl)sulfonamido)-6-(2-((tert-butyldimethylsilyl)oxy)ethyl)-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate(1.79 g, 2.53 mmol) in DCM (8.5 mL). After stirring at rt for 18 h, itwas diluted with DCM and quenched with saturated NaHCO₃. Upon cessationof bubbling, the layers were separated and the aqueous was extractedwith DCM (3×). The combined organic layers were dried over Na₂SO₄,filtered and concentrated in vacuo, resulting in a white foam. LCMS:R_(t)=0.94 min; m/z=605.3 (M+1) Method 2m_acidic.

Step 5:(3R,4aS,7aS)-3-((benzyloxy)amino)-6-(2-((tert-butyldimethylsilyl)oxy)ethyl)octahydro-7H-pyrrolo[3,4-b]pyridin-7-one.To a slurry ofN-(benzyloxy)-N-((3R,4aS,7aS)-6-(2-((tert-butyldimethylsilyl)oxy)ethyl)-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridin-3-yl)-2-nitrobenzenesulfonamide(1.53 g, 2.53 mmol) and K₂CO₃ (1.753 g, 12.68 mmol) in ACN (25 mL) wasadded thiophenol (1.343 ml, 12.65 mmol). After 22 h it was filtered andconcentrated in vacuo. The crude residue was purified via silica gelchromatography, affording the title compound (919 mg, 87%, 2-steps) asan off-white foam. LCMS: R_(t)=0.82 min; m/z=420.4 (M+1) Method2m_acidic.

Step 6:(4R,5aS,8aS)-3-(benzyloxy)-7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione.To a solution of(3R,4aS,7aS)-3-((benzyloxy)amino)-6-(2-((tert-butyldimethylsilyl)oxy)ethyl)octahydro-7H-pyrrolo[3,4-b]pyridin-7-one(919 mg, 2.19 mmol) and DIPEA (1.2 mL, 6.87 mmol) in acetonitrile (68.4mL) at 0° C. was added phosgene (15-20% in toluene, 1.60 mL, 2.24 mmol)as a solution in acetonitrile (10 mL) at a rate of 8 mL/h. It wasallowed to slowly raise to rt. After 20 h it was concentrated in vacuo,partitioned between EtOAc/HCl (aq, 0.2 M) and the phases were separated.The aqueous layer was extracted with EtOAc (2×) and the combined organiclayers were washed with brine, saturated NaHCO₃, dried overNa₂SO₄/MgSO₄, filtered and concentrated in vacuo. The crude residue waspurified via silica gel chromatography, affording the title compound(549 mg, 56%) as a white foam. LCMS: R_(t)=0.95 min; m/z=446.4 (M+1)Method 2m_acidic.

Step 7:(4R,5aS,8aS)-7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-hydroxyhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione.A slurry of(4R,5aS,8aS)-3-(benzyloxy)-7-(2-((tert-butyldimethylsilyl)oxy)ethyl)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione(110 mg, 0.247 mmol) and Pd—C (10% Degussa type 101, 50% water, 25 mg,0.012 mmol) in MeOH (2.5 mL) was degassed and backfilled with H₂ (3×).After 3 h of vigorous stirring the slurry was purged with N₂, filteredthrough celite and concentrated in vacuo. Toluene was added and it wassonicated then reconcentrated. Assumed quantitative yield. LCMS:R_(t)=0.71 min; m/z=356.4 (M+1) Method 2m_acidic.

Step 8: Tetrabutylammonium(4R,5aS,8aS)-7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. To a solution of(4R,5aS,8aS)-7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-hydroxyhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione(0.247 mmol) in pyridine (1.6 mL) was added SO₃.Py (197 mg, 1.24 mmol).After stirring for 17 h at rt the mixture was concentrated in vacuo andslurried in DCM then filtered and reconcentrated in vacuo. The resultingsolid was dissolved in NaH₂PO₄ (1 M aq, 20 mL), whereupontetrabutylammonium hydrogen sulfate (131 mg, 0.386 mmol) was added.After stirring for 45 min it was extracted with DCM (4×) and thecombined organic layers were dried over Na₂SO₄, filtered andconcentrated in vacuo. The crude residue was purified via silica gelchromatography (MeOH-DCM, 0-20%) to afford the title compound (56 mg,34%, 3-steps) as an off-white foam. LCMS: R_(t)=0.81 min; m/z=436.3(M+1) Method 2m_acidic.

Step 9: Sodium(4R,5aS,8aS)-7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. DOWEX 50Wx8 hydrogen form 200-400 mesh was conditioned bystirring with NaOH (2 N) for 3 h. The resin was loaded onto a glasscolumn and washed with water (until pH=6) followed by water:acetone(1:1). A solution of tetrabutylammonium(4R,5aS,8aS)-7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate (56 mg, 0.083 mmol) in water:acetone (1:1) was loaded onto andpassed through the column, eluting with water:acetone (1:1). The samplewas concentrated in vacuo (bath temp <30° C.) and lyophilized, affordingthe title compound (36 mg, 95% Yield) as a white powder. LCMS:R_(t)=0.84 min; m/z=436.3 (M+1) Method 2m_acidic.

Step 10: Sodium(4R,5aS,8aS)-7-(2-hydroxyethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. To a slurry of sodium(4R,5aS,8aS)-7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate (36 mg, 0.079 mmol) in acetonitrile (790 μL) was addedtriethylamine trihydrofluoride (13.07 μl, 0.079 mmol), drop-wise and theresulting solution was heated to 45° C. for 3 h. Additionaltriethylamine trihydrofluoride (13.07 μl, 0.079 mmol) was added and itwas heated to 45° C. for 2 h then concentrated in vacuo. The cruderesidue was taken up in phosphate buffer (pH=6) and purified by reversephase prep HPLC (T3, Atlantis column, 30×100 mm, 5 Lm, C18 column;ACN-water with 3.75 mmol NH₄OAC buffer, 20-60 mL/min), affording thetitle compound (13.9 mg) as a white powder. LCMS: R_(t)=0.34 min;m/z=322.2 (M+1) Method T3_3m_polar. ¹H NMR (500 MHz, D₂O) δ 4.8 (d,J=8.0 Hz, 1H), 4.14 (s, 1H), 3.69 (t, J=5.4 Hz, 2H), 3.61 (dd, J=10.7,6.3 Hz, 1H), 3.49-3.37 (m, 2H), 3.29-3.22 (m, 1H), 3.17-3.14 (m, 1H),2.84 (d, J=12.2 Hz, 1H), 2.75-2.67 (m, 1H), 2.45 (ddt, J=14.7, 8.7, 3.0Hz, 1H), 1.54 (ddd, J=14.8, 9.0, 2.0 Hz, 1H).

EXAMPLE 5(4R,5aS,8aS)-7-(2-aminoethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylhydrogen sulfate.

Step 1:(4R,5aS,8aS)-3-(benzyloxy)-7-(2-hydroxyethyl)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione.To a soln of(4R,5aS,8aS)-3-(benzyloxy)-7-(2-((tert-butyldimethylsilyl)oxy)ethyl)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione(530 mg, 1.19 mmol) in THF (12 mL) at 0° C. was added TBAF (1.2 mL, 1.20mmol). After 1 h at 0° C. it was concentrated in vacuo, partitionedbetween EtOAc/water and the phases were separated. The aqueous layer wasextracted with EtOAc (2×) and the combined organic layers were washedwith brine, dried over Na₂SO₄/MgSO₄, filtered and concentrated in vacuo.The crude residue was purified by silica gel chromatography (MeOH-DCM,0-7%) to afford the title compound (268 mg, 68%) as a white solid. LCMS:R_(t)=0.55 min; m/z=332.3 (M+1) Method 2m_acidic. ¹H NMR (500 MHz,CDCl₃-d) δ 7.48-7.36 (m, 5H), 5.09 (d, J=11.3 Hz, 1H), 4.93 (d, J=11.2Hz, 1H), 4.18 (d, J=7.8 Hz, 1H), 3.87-3.75 (m, 2H), 3.66-3.54 (m, 2H),3.42-3.35 (m, 1H), 3.30 (s, 1H), 3.10 (d, J=12.2 Hz, 1H), 3.03 (d,J=10.1 Hz, 1H), 2.81-2.72 (m, 2H), 2.48-2.39 (m, 2H), 1.38 (dd, J=14.2,9.2 Hz, 1H).

Step 2:2-((4R,5aS,8aS)-3-(benzyloxy)-2,8-dioxooctahydro-7H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-7-yl)ethylmethanesulfonate. To a soln of(4R,5aS,8aS)-3-(benzyloxy)-7-(2-hydroxyethyl)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione(265.4 mg, 0.801 mmol) and TEA (140 μl, 1.00 mmol) in DCM (4.0 mL) wasadded MsCl (65.5 μl, 0.841 mmol). After 45 min it was washed with water.The aqeuous layer was extracted with DCM (2×) and the combined organiclayers were dried over Na₂SO₄/MgSO₄, filtered and concd in vacuo,affording the title compound (332 mg) as a white solid. LCMS: R_(t)=0.55min; m/z=410.3 (M+1) Method 2m_acidic.

Step 3: Di-tert-butyl(2-((4R,5aS,8aS)-3-(benzyloxy)-2,8-dioxooctahydro-7H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-7-yl)ethyl)iminodicarboxylate.To a soln of di-tert-butyl-iminodicarboxylate (153 mg, 0.704 mmol) inDMF (3.2 mL) was added potassium tert-butoxide (1 M in THF, 700 μL,0.700 mmol). After 30 min at rt, a solution of2-((4R,5aS,8aS)-3-(benzyloxy)-2,8-dioxooctahydro-7H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-7-yl)ethylmethanesulfonate (262 mg, 0.640 mmol) in DMF (2 mL, 2×500 μL washes) wasadded. It was stirred at rt for 10 min, heated to 50° C. for 100 minthen stirred at rt for 12 h, whereupon it was diluted with EtOAc andwashed with brine (saturated). The aqueous layer was extracted withEtOAc (2×) and the combined organic layers were dried over Na₂SO₄/MgSO₄,filtered and concentrated in vacuo. The crude residue was purified viasilica get chromatography (EtOAc-heptane, 0-90%), affording the titlecompound (293 mg, 86%) as a white solid. LCMS: R_(t)=0.87 min; m/z=431.4(M-Boc+1) Method 2m_acidic.

Step 4: tert-butyl(2-((4R,5aS,8aS)-3-hydroxy-2,8-dioxooctahydro-7H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-7-yl)ethyl)carbamate.A slurry of di-tert-butyl(2-((4R,5aS,8aS)-3-(benzyloxy)-2,8-dioxooctahydro-7H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-7-yl)ethyl)iminodicarboxylate(226.8 mg, 0.427 mmol) and Pd—C (10% Degussa type 101, 50% water, 45.3mg, 0.021 mmol) in MeOH (4.2 mL) was degassed and backfilled with H₂(3×). After 3 h of vigorous stirring the slurry was purged with N₂,filtered through celite and concentrated in vacuo. Toluene was added andit was sonicated then reconcentrated. Assumed quantitative yield. LCMS:R_(t)=0.65 min; m/z=341.4 (M+1) Method 2m_acidic.

Step 5: Pyridin-1-ium(4R,5aS,8aS)-7-(2-((tert-butoxycarbonyl)amino)ethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate

To a solution of tert-butyl(2-((4R,5aS,8aS)-3-hydroxy-2,8-dioxooctahydro-7H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-7-yl)ethyl)carbamateinpyridine (3 mL) was added SO₃.Pyridine (335 mg, 2.10 mmol). After 15 hat rt it was concentrated in vacuo, slurried in DCM and filtered,affording the title compound as an off-white solid. Assumed quantitativeyield. LCMS: R_(t)=0.59 min; m/z=321.4 (M-Boc+1) Method 2m_acidic.

Step 6:(4R,5aS,8aS)-7-(2-aminoethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylhydrogen sulfate. To a slurry of pyridin-1-ium(4R,5aS,8aS)-7-(2-((tert-butoxycarbonyl)amino)ethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate (210 mg, 0.421 mmol) in DCM (4.2 mL) at 0° C. was added TFA (973μl, 12.63 mmol). After 2.5 h at 0° C. it was concentrated in vacuo,slurried in DCM and reconcentrated. The crude residue was taken up inphosphate buffer (pH=6), filtered and purified by reverse phase prepHPLC (T3, Atlantis column, 30×100 mm, 5 Lm, C18 column; water with 3.75mmol NH₄OAC buffer, 20-60 mL/min), affording the title compound (155 mg)as a white powder. LCMS: R_(t)=0.22 min; m/z=321.4 (M+1) MethodT33m_polar. ¹H NMR (500 MHz, D₂O) δ 4.00 (d, J=8.1 Hz, 1H), 3.94 (s,1H), 3.61 (dt, J=14.3, 6.9 Hz, 1H), 3.45 (dd, J=10.5, 6.4 Hz, 1H), 3.28(dt, J=14.8, 5.6 Hz, 1H), 3.06 (brd, J=12.4 Hz, 1H), 2.96 (t, J=6.2 Hz,2H), 2.92 (d, J=10.5 Hz, 1H), 2.68 (d, J=12.3 Hz, 1H), 2.55 (p, J=8.3Hz, 1H), 2.31-2.21 (m, 1H), 1.38 (dd, J=14.9, 9.1 Hz, 1H).

EXAMPLE 6 Sodium(4R,5aS,8aS)-2,8-dioxo-7-propylhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate.

Step 1: tert-Butyl(3R,4aS,7aS)-6-allyl-3-((N-(benzyloxy)-2-nitrophenyl)sulfonamido)-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate.Toa solution of tert-butyl(3S,4aS,7aS)-6-allyl-3-hydroxy-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate(786.4 mg, 2.65 mmol), N-(benzyloxy)-2-nitrobenzenesulfonamide (900 mg,2.92 mmol) and triphenylphosphine (835 mg, 3.18 mmol) in THF (29 mL) at−17° C. was added DIAD (0.640 mL, 3.18 mmol) as a soln in THF (4.1 mL)drop-wise at 6:30 μm. It was allowed to slowly warm to rt and stir for22 h then concentrated in vacuo and purified directly via silica gelchromatography (EtOAc-heptane, 0-40%), affording the title compound (846mg, 54%) as an off-white solid. LCMS: R_(t)=0.95 min; m/z=587.3 (M+1)Method 2m_acidic.

Step 2:N-((3R,4aS,7aS)-6-allyl-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridin-3-yl)-N-(benzyloxy)-2-nitrobenzenesulfonamide.To a flask charged with zinc(II) bromide (681 mg, 3.02 mmol, dried at200° C. for 4 h) and tert-butyl(3R,4aS,7aS)-6-allyl-3-((N-(benzyloxy)-2-nitrophenyl)sulfonamido)-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate(845 mg, 1.44 mmol), under N₂, was added DCM (4.8 mL). After stirring atrt for 15 h, it was diluted with DCM and quenched with saturated NaHCO₃.Upon cessation of bubbling, the layers were separated and the aqueouswas extracted with DCM (3×). The combined organic layers were dried overNa₂SO₄ and concentrated in vacuo, resulting in a white foam. Assumedquantitative yield. LCMS: Rt=0.70 min; m/z=487.2 (M+1) Method 2m_acidic.

Step 3:(3R,4aS,7aS)-6-allyl-3-((benzyloxy)amino)octahydro-7H-pyrrolo[3,4-b]pyridin-7-one.To a slurry ofN-((3R,4aS,7aS)-6-allyl-7-oxooctahydro-1H-pyrrolo[3,4-b]pyridin-3-yl)-N-(benzyloxy)-2-nitrobenzenesulfonamide(1.44 mmol) and K₂CO₃ (995 mg, 7.20 mmol) in ACN (14.4 mL) was addedthiophenol (764 μL, 7.20 mmol). After 21 h it was filtered andconcentrated in vacuo. The crude residue was purified via silica gelchromatography (MeOH-DCM, 0-8%), affording the title compound (385 mg,89%, 2-steps) as an off-white foam. LCMS: Rt=0.49 min; m/z=302.4 (M+1)Method 2m_acidic.

Step 4:(4R,5aS,8aS)-7-allyl-3-(benzyloxy)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione.To a solution of(3R,4aS,7aS)-6-allyl-3-((benzyloxy)amino)octahydro-7H-pyrrolo[3,4-b]pyridin-7-one(385 mg, 1.28 mmol) and DIPEA (670 μL, 3.83 mmol) in ACN (40 mL) at 0°C. was added phosgene (1.20 mL, 1.66 mmol) as a solution in ACN (5.7 mL)at a rate of 6.5 mL/h. It was allowed to slowly raise to rt. After 20 hit was concentrated in vacuo, partitioned between EtOAc/HCl (0.2 N) andthe phases were separated. The aqueous layer was extracted with EtOAc(3×) and the combined organic layers were washed with brine andsaturated NaHCO₃. The brine wash was combined with the saturated NaHCO₃wash and the solution was extracted with 10% MeOH/DCM (2×). The acidicaqueous layer was re-extracted with 10% MeOH/DCM (2×) and the combinedorganic layers were dried over Na₂SO₄, filtered and concentrated invacuo. The crude residue was purified via silica gel chromatography(MeOH-DCM, 0-10%), affording the title compound (369.2 mg, 88%) as awhite foam. LCMS: Rt=0.60 min; m/z=328.4 (M+1) Method 2m_acidic.

Step 5:(4R,5aS,8aS)-3-hydroxy-7-propylhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione.A slurry of(4R,5aS,8aS)-7-allyl-3-(benzyloxy)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione(209 mg, 0.638 mmol) and Pd—C (10% Degussa type 101, 50% water, 41 mg,0.019 mmol) in MeOH (6.4 mL) was degassed and backfilled with H₂ (3×).After 5 h of vigorous stirring the slurry was purged with N₂, more Pd—C(10% Degussa type 101, 50% water, 41 mg, 0.019 mmol) was added and itwas degassed and backfilled with H₂ (3×). After 2 h of vigorous stirringit was purged with N₂ then filtered through celite and concentrated invacuo. Toluene was added and it was sonicated then reconcentrated.Assumed quantitative yield. LCMS: R_(t)=0.27 min; m/z=240.3 (M+1) Method2m_acidic.

Step 6: Tetrabutylammonium(4R,5aS,8aS)-2,8-dioxo-7-propylhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. To a solution of(4R,5aS,8aS)-3-hydroxy-7-propylhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione(0.638 mmol) in pyridine (6.4 mL) was added SO₃.Py (508 mg, 3.19 mmol).After stirring for 13 h at rt the mixture was filtered and concentratedin vacuo. The resulting solid was dissolved in NaH₂PO₄ (1 M aq, 40 mL),whereupon tetrabutylammonium hydrogen sulfate (325 mg, 0.957 mmol) wasadded. After stirring for 1.5 h it was extracted with DCM (4×) and thecombined organic layers were dried over Na₂SO₄, filtered andconcentrated in vacuo. The crude residue was purified via silica gelchromatography (MeOH-DCM, 0-15%) to afford the title compound (229 mg,64%, 3-steps) as a white solid. LCMS: R_(t)=0.81 min; m/z=436.3 (M+1)Method 2m_acidic.

Step 7: Sodium(4R,5aS,8aS)-2,8-dioxo-7-propylhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. DOWEX 50Wx8 hydrogen form 200-400 mesh was conditioned bystirring with NaOH (2 N) for 2 h. The resin was loaded onto a glasscolumn and washed with water (until pH=6) followed by water:acetone(1:1). A solution of tetrabutylammonium(4R,5aS,8aS)-2,8-dioxo-7-propylhexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate (228 mg, 0.408 mmol) in water:acetone (1:1) was loaded onto andpassed through the column, eluting with water:acetone (1:1). The samplewas concentrated in vacuo (bath temp <30° C.) and lyophilized, affordingthe title compound (120 mg, 85%) as a white powder. LCMS: R_(t)=0.30min; m/z=320.3 (M+1) Method 2m_acidic. ¹H NMR (500 MHz, D₂O) δ 4.18 (d,J=8.0 Hz, 1H), 4.14 (s, 1H), 3.54 (dd, J=10.9, 6.3 Hz, 1H), 3.32-3.17(m, 3H), 3.10 (d, J=10.8 Hz, 1H), 2.80 (d, J=12.2 Hz, 1H), 2.69 (p,J=8.2 Hz, 1H), 2.50-2.41 (m, 1H), 1.57-1.47 (m, 3H), 0.81 (t, J=7.4,3H).

EXAMPLE 7 Sodium(4R,5aR,9aS)-2,9-dioxooctahydro-1,4-methanopyrido[3,4-d][1,3]diazepin-3(2H)-ylhydrogen sulfate.

Step 1: 1-(tert-butyl) 2-ethyl(2S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)piperidine-1,2-dicarboxylate.To a suspension of (2S,5R)-ethyl5-((benzyloxy)amino)piperidine-2-carboxylate oxalate (13.25 g, 36.0mmol) in EtOAc (200 mL) were added Na₂CO₃ (2.0 M, 80 mL, 160 mmol) andsodium hydroxide (1.0 M, 40 mL, 40 mmol). The mixture was stirred atroom temperature for 30 min. The precipitate formed was filtered off andthe two layers of the filtrate were separated. The organic layer waswashed with brine (50 ml), dried over Na₂SO₄, filtered and concentratedin vacuo, affording a viscous oil (10.0 g)

To a solution of this oil (10.0 g, 35.9 mmol) in THF (100 ml) were addedBoc-anhydride (23.5 g, 108 mmol), triethylamine (15.0 ml, 108 mmol) andDMAP (4.38 g, 35.9 mmol). The reaction mixture was stirred for 60 h andthen heated at 50° C. for 2 days. The solvent was removed in vacuo andtaken back up in EtOAc/Heptane (300 mL, 1/1), washed with water (100mL), HCl (0.1 N, 50 mL), brine (50 mL), dried over Na₂SO₄, filtered andconcentrated in vacuo. The crude residue was purified via silica gelchromatography (EtOAc-Heptane, 0-40%) to afford the title compound (9.6g, 55%) as an oil. LCMS: R_(t)=1.19 min, m/z=479.2 (M+1), Method2m_acidic.

Step 2:(2S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)-1-(tert-butoxycarbonyl)piperidine-2-carboxylicacid. To a solution of 1-(tert-butyl) 2-ethyl(2S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)piperidine-1,2-dicarboxylate(9.60 mg, 20.06 mmol) in THF:MeOH (3:1, 80 mL) at 0° C. was slowly addeda solution of sodium hydroxide (1 N, 40 mL). After 5 h at rt, HCl (1 N,41 mL) was slowly added the it was extracted with EtOAc (300 mL). Theorganic layer was dried over Na₂SO₄, filtered and concentrated in vacuoto afford the title compound (8.78 g, 97%) as a soft solid. LCMS:R_(t)=1.05 min, m/z=451.2 (M+1) Method 2m_acidic.

Step 3: tert-Butyl(2S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)-2-(quinolin-8-ylcarbamoyl)piperidine-1-carboxylate.To a solution of(2S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)-1-(tert-butoxycarbonyl)piperidine-2-carboxylicacid (6.010 g, 13.34 mmol) in DCM (100 mL) at 0° C. was addedquinolin-8-amine (2116 mg, 14.67 mmol) followed by DIPEA (4.66 mL, 26.7mmol) and HATU (6.087 g, 16.01 mmol). After stirring under argon at rtfor 2.5 h, the mixture was poured into water (150 mL) and extracted withDCM (100 mL). The organic layer was dried over MgSO₄, filtered andconcentrated in vacuo. The crude residue was purified via silica gelchromatography (EtOAc-Heptane, 10-40%), affording the title compound(6.60 g, 86%) as a soft solid. LCMS: R_(t)=1.22 min, m/z=577.3 (M+1),Method 2m_acidic.

Step 4: tert-Butyl(2S,3S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)-3-(2-methoxy-2-oxoethyl)-2-(quinolin-8-ylcarbamoyl)piperidine-1-carboxylate.To a solution of tert-butyl(2S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)-2-(quinolin-8-ylcarbamoyl)piperidine-1-carboxylate(5.580 g, 9.68 mmol) in 2-methyl-2-butanol (95 mL) were added dibenzylhydrogen phosphate (538 mg, 1.94 mmol), silver carbonate (5.336 mg,19.35 mmol), Pd(II) acetate (434 mg, 1.94 mmol) and methyl2-bromoacetate (2.83 mL, 29.0 mmol). The mixture was purged with argon,sealed and heated to 110° C. for 20 h. Additional Pd(II) acetate (217mg, 0.97 mmol) and methyl 2-bromoacetate (1.88 mL, 19.36 mmol) wereadded and the reaction mixture was stirred at 110° C. for another 20 h.The mixture was cooled to room temperature, diluted with DCM (100 ml),filtered and concentrated in vacuo. The crude residue was purified viasilica gel chromatography (EtOAc-Heptane, 0-35%) to afford the titlecompound (2.170 g, 35%) as a viscous oil. LCMS: R_(t)=1.26 min,m/z=649.3 (M+1), Method 2m_acidic.

Step 5: tert-Butyl(2S,3S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)-3-(2-hydroxyethyl)-2-(quinolin-8-ylcarbamoyl)piperidine-1-carboxylate.To a solution of tert-butyl(2S,3S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)-3-(2-methoxy-2-oxoethyl)-2-(quinolin-8-ylcarbamoyl)piperidine-1-carboxylate(2.40 g, 3.70 mmol) in THF (60 mL) at 0° C. was added super-hydride (1.0M in THF, 18.50 mL, 18.5 mmol). After stirring at 0° C. for 5 h, AcOH(50% aq, 10 ml) was added followed by saturated NH₄Cl (30 mL) and EtOAc(150 mL). The layers were separated and the organic layer was washedwith brine (50 mL), dried over Na₂SO₄, filtered and concentrated invacuo. The crude residue was purified via silica gel chromatography(EtOAc-Heptane, 10-60%) to afford the title compound (680 mg, 30%).LCMS: R_(t)=1.16 min, m/z=621.1 (M+1) Method 2m_acidic.

Step 6: tert-Butyl(2S,3S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)-3-(2-((methylsulfonyl)oxy)ethyl)-2-(quinolin-8-ylcarbamoyl)piperidine-1-carboxylate.To a solution of tert-butyl(2S,3S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)-3-(2-hydroxyethyl)-2-(quinolin-8-ylcarbamoyl)piperidine-1-carboxylate(680 mg, 1.10 mmol) in dichloromethane (20 mL) at 0° C. were addedtriethylamine (0.30 mL, 2.19 mmol) and methylsulfonyl chloride (0.17 mL,2.19 mmol). After stirring for 20 h at rt the mixture was diluted withwater (20 mL) and EtOAc (100 mL) and stirred for an additional 15 min,whereupon the layers were separated. The organic layer was washed withNaH₂PO₄ (1.0 M, 2×40 mL), brine (30 mL), dried over Na₂SO₄, filtered andconcentrated in vacuo, affording the title compound (quantitative yield)as a soft solid. LCMS: R_(t)=1.22 min, m/z=699.4 (M+1), Method2m_acidic.

Step 7: tert-Butyl(3R,4aR,8aS)-3-((benzyloxy)(tert-butoxycarbonyl)amino)-8-oxo-7-(quinolin-8-yl)octahydro-1,7-naphthyridine-1(2H)-carboxylate.To a solution of tert-butyl(2S,3S,5R)-5-((benzyloxy)(tert-butoxycarbonyl)amino)-3-(2-((methylsulfonyl)oxy)ethyl)-2-(quinolin-8-ylcarbamoyl)piperidine-1-carboxylate(690 mg, 0.99 mmol) in THF (16 mL) at 0° C. was added LDA (1.0 M inTHF/hexane, 1.97 mL). After stirring at 0° C. for 2.5 h, it was warmedto rt and stirred overnight. Saturated NH₄Cl (20 mL) solution was addedand the mixture was extracted with EtOAc (80 mL). The organic layer waswashed with brine (20 mL), dried over Na₂SO₄, filtered and concentratedin vacuo. The crude residue was purified via silica gel chromatography(EtOAc-Heptane, 30-80%) to afford the title compound (680 mg, 45%) as asoft solid. LCMS: R_(t)=1.03 min, m/z=603.4 (M+1), Method 2m_acidic.

Step 8: tert-Butyl(3R,4aR,8aS)-3-((benzyloxy)(tert-butoxycarbonyl)amino)-8-oxooctahydro-1,7-naphthyridine-1(2H)-carboxylate.A solution of tert-butyl(3R,4aR,8aS)-3-((benzyloxy)(tert-butoxycarbonyl)amino)-8-oxo-7-(quinolin-8-yl)octahydro-1,7-naphthyridine-1(2H)-carboxylate (270 mg, 0.448 mmol) in dry DCM (15 mL) at −78° C. wassparged with O₃ until a blue color persisted, whereupon the spargingline was removed. After stirring at −78° C. for 45 min, the blue colordisappeared and it was again sparged with O₃ until the blue colorpersisted. After 15 min of stirring, the system was sparged with O₂until it remained colorless. To the solution was added dimethyl sulfide(100 μL, 1.36 mmol). After stirring at room temperature for 1 h, themixture was concentrated in vacuo. The residue was redissolved in THF (5mL) and NH₄OH (25% aq, 5 mL) was added. After stirring for 16 h, themixture was diluted with EtOAc (50 mL) and the organic layer was washedwith water (20 ml), brine (20 ml), dried over Na₂SO₄, filtered andconcentrated to in vacuo. The crude residue was purified via silica gelchromatography (EtOAc-Heptane, 70-100%) to afford the title compound (96mg, 45%) as a solid. LCMS: R_(t)=1.00 min, m/z=476.2 (M+1), Method2m_acidic.

Step 9:(3R,4aR,8aS)-3-((benzyloxy)amino)octahydro-1,7-naphthyridin-8(2H)-one.To a solution of tert-butyl(3R,4aR,8aS)-3-((benzyloxy)(tert-butoxycarbonyl)amino)-8-oxooctahydro-1,7-naphthyridine-1(2H)-carboxylate (120 mg, 0.252 mmol) in DCM (3 mL) at 0° C. was slowlyadded TFA (1.5 mL). After 3 h at 0° C. then rt for 1 h, it wasconcentrated in vacuo (bath temp <30° C.). The residue was taken up inDCM:EtOH (5:1, 30 mL) and Na₂CO₃ (2 M, 10 mL) was added. The layers wereseparated and the aqueous layer was extracted with DCM:EtOH (5:1, 2×30mL). The combined organic layers were dried over Na₂SO₄, filtered andconcentrated in vacuo. The crude residue was purified via silica gelchromatography (MeOH-DCM, 10-25%) to afford the title compound (60 mg,86%) as a solid. LCMS: R_(t)=0.58 min, m/z=276.1 (M+1), Method2m_acidic.

Step 10:(4R,5aR,9aS)-3-(benzyloxy)hexahydro-1,4-methanopyrido[3,4-d][1,3]diazepine-2,9(3H,6H)-dione.To a solution of(3R,4aR,8aS)-3-((benzyloxy)amino)octahydro-1,7-naphthyridin-8(2H)-one(56 mg, 0.20 mmol) in ACN (21 mL) at 0° C. under N₂ was added DIPEA (140μL, 0.81 mmol). A solution of triphosgene (24 mg, 0.08 mmol) in ACN (3mL) was added via syringe pump (0.1 mL/min). After stirring at 0° C. for6 h it was partially concentrated (˜10 mL) in vacuo, diluted with DCM(40 mL), washed with water (20 mL), brine (20 mL), dried over Na₂SO₄,filtered and concentrated in vacuo. The crude residue was purified viasilica gel chromatography (MeOH-DCM, 0-5%) to afford the title compound(50 mg, 82%) as an off-white solid. LCMS: R_(t)=0.65 min, m/z=302.0(M+1), Method 2m_acidic.

Step 11:(4R,5aR,9aS)-3-hydroxyhexahydro-1,4-methanopyrido[3,4-d][1,3]diazepine-2,9(3H,6H)-dione. A slurry of(4R,5aR,9aS)-3-(benzyloxy)hexahydro-1,4-methanopyrido[3,4-d][1,3]diazepine-2,9(3H,6H)-dione(50 mg, 0.17 mmol) and Pd—C (10% Degussa type 101, 50% water, 27 mg) inMeOH:DCM (3:1, 4 mL) was evacuated and backfilled with H₂. After 2 h ofvigorous stirring, it was filtered through a plug of celite, washingwith MeOH, and concentrated in vacuo. LCMS: R_(t)=0.20 min, m/z=212.0(M+1) Method 2m_acidic.

Step 12: Tetrabutylammonium(4R,5aR,9aS)-2,9-dioxooctahydro-1,4-methanopyrido[3,4-d][1,3]diazepin-3(2H)-ylsulfate. To a slurry of crude(4R,5aR,9aS)-3-hydroxyhexahydro-1,4-methanopyrido[3,4-d][1,3]diazepine-2,9(3H,6H)-dione(35 mg, 0.17 mmol) in pyridine (3 ml) at 0° C. was added SO₃.Py (132 mg,0.83 mmol). After vigorous stirring at rt for 20 h, the slurry wasfiltered and the solid was washed with cold DCM (5 mL). The filtrate wasconcentrated in vacuo (bath temp <30° C.) and the crude residue wasdissolved in NaH₂PO₄ (1 M, 10 mL), whereupon tetrabutylammonium hydrogensulfate (84 mg, 0.25 mmol) was added. After 30 min it was extracted withCHCl₃:IPA (4:1, 3×30 mL). The combined organic layers were dried overNa₂SO₄, filtered and concentrated in vacuo. The crude residue waspurified via silica gel chromatography (MeOH-DCM, 5-20%) to afford thetitle compound as a white foam. LCMS: R_(t)=0.15 min, m/z=292.0 (M+1),Method 2m_acidic.

Step 13: Sodium(4R,5aR,9aS)-2,9-dioxooctahydro-1,4-methanopyrido[3,4-d][1,3]diazepin-3(2H)-ylhydrogen sulfate. DOWEX 50Wx8 hydrogen form 200-400 mesh was conditionedby stirring with NaOH (2 N) for 2 h. The resin was loaded onto a glasscolumn and washed with water (until pH=6) followed by water:acetone(1:1). Tetrabutylammonium(4R,5aR,9aS)-2,9-dioxooctahydro-1,4-methanopyrido[3,4-d][1,3]diazepin-3(2H)-ylsulfate (228 mg, 0.408 mmol) in acetone:water (1:1) was loaded onto andpassed through the column, eluting with water (20 ml) then acetone:water(1:4, 30 ml). The sample was lyophilized to afford the title compound(28 mg, 52%) as a white solid. LCMS: R_(t)=0.29 min, m/z=291.8 (M+1)Method T3_3m_polar; ¹H NMR (500 MHz, D₂O) δ 4.31 (m, 1H), 4.09 (d, J=7.1Hz, 1H), 3.55 (td, J=12.8, 4.3 Hz, 1H), 3.26-3.34 (m, 2H), 2.87 (d,J=12.3 Hz, 1H), 2.55-2.64 (m, 1H), 2.21-2.30 (m, 1H), 1.99-2.09 (m, 1H),1.80-1.88 (m, 1H) 1.72-1.79 (m, 1H).

EXAMPLE 8 Sodium(4R,5aS,6R,8aS)-6-(methoxymethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylhydrogen sulfate.

Step 1: Methyl(5R)-6-(benzyloxy)-3-(1-((tert-butoxycarbonyl)amino)-2-methoxyethyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate(Mixture of Diastereoisomers).

Intermediate C (1.10 g, 3.82 mmol), Boc-L-Ser(OMe)-OH (1.04 g, 4.58mmol) and Ir[df(CF₃)ppy₂(dtbpy)]PF₆ (43 mg, 0.04 mmol) were dissolved inDMF (16 mL). To the solution was added finely ground potassium phosphatedibasic (0.62 g, 4.58 mmol) and the resulting suspension was stirred andirradiated for 12 days with a Kessil H150-Blue lamp from a distance of<2 cm. After 3 and after 9 days Ir[df(CF₃)ppy₂(dtbpy)]PF₆ (43 mg, 0.04mmol) was added (total of 3 mol % catalyst). To the reaction mixture wasadded water (15 mL) followed by saturated NaHCO₃ (aq, 15 mL), which wasthen extracted with TBME (3×60 mL). The combined organic phases werewashed with brine (10 mL), dried over Na₂SO₄ and concentrated in vacuo,resulting in the title compound (1.85 g) as yellow oil consisting of 4diastereoisomers (ratio 39:16:11:34). LCMS: R_(t)=1.02 min, 1.05 min,1.08 min, 1.12 min all with m/z=464 (M+1), LCMS_2MIN_REACTION_MONITORING.

Step 2:(4R,5aS,6R,8aS)-3-(benzyloxy)-6-(methoxymethyl)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(3H)-dione.

To a solution of methyl(5R)-6-(benzyloxy)-3-(1-((tert-butoxycarbonyl)amino)-2-methoxyethyl)-7-oxo-1,6-diazabicyclo[3.2.1]octane-2-carboxylate(1.85 g, 4.0 mmol) in DCM (60 mL) at 0° C. was added TFA (15.4 mL, 200mmol), drop-wise. The reaction mixture was stirred at rt for 1.5 h, thenconcentrated in vacuo. The crude residue was dissolved in DCM (60 mL)then triethylamine (11.1 mL, 80 mmol) was added, drop-wise. The reactionmixture stirred at rt overnight, whereupon it was concentrated tofurnish a reddish oil (6.8 g). Water (20 mL) was added and the mixtureextracted with TBME (3×80 mL). The combined organic phases were driedover Na₂SO₄ and concentrated in vacuo to yield a yellow oil (1.13 g).The aqueous phase was saturated with NaCl (s) and further extracted withDCM (3×80 mL). The combined organic phases were dried over Na₂SO₄ andconcentrated in vacuo to yield additional crude product (0.95 g). Thecombined crude product was purified by HPLC chromatography (Sunfire-C18,5 um, 50×250 mm, water/ACN+0.1% TFA, 100 ml/min, 18-38% over 21 min,total 35 min) where the pH of the fractions was adjusted to 6.9 viaaddition of saturated NaHCO₃ (aq) and lyophilized to afford a lightbrown residue (0.59 g). This residue was dissolved in ACN/water andpurified over a C18 cartridge (ACN-water), whereupon the lyophilizedmaterial afforded the title compound (62 mg, 4.1% 3-steps). LCMS:R_(t)=0.70 min, m/z=332 (M+1), LCMS_2 MIN_REACTION_MONITORING.

Step 3:(4R,5aS,6R,8aS)-3-hydroxy-6-(methoxymethyl)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione.(4R,5aS,6R,8aS)-3-(benzyloxy)-6-(methoxymethyl)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione(59 mg, 0.178 mmol) was dissolved in MeOH:DCM (1:1, 1.78 mL). Themixture was purged with nitrogen, Pd—C (10% Degussa type, 101, 50%water, 37.9 mg, 0.018 mmol) was added, then left under an H₂ atmosphereat for 90 min. The mixture was filtered through celite, eluting withDCM:MeOH (1:1) and concentrated in vacuo to afford the title compound(49 mg, quantitative) as a colorless solid. LC/MS: R_(t)=0.12 min;m/z=242.0 (M+1) Method 2m_acidic.

Step 4: tetrabutylammonium(4R,5aS,6R,8aS)-6-(methoxymethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate. To a solution of(4R,5aS,6R,8aS)-3-hydroxy-6-(methoxymethyl)hexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepine-2,8(8aH)-dione(42 mg, 0.174 mmol) in pyridine (1.85 mL) was added SO₃.pyridine (139mg, 0.870 mmol). The mixture was stirred for 18 h, then filtered througha membrane filter and concentrated in vacuo (bath temp <30° C.). Thecrude residue was dissolved in saturated NaH₂PO₄ and washed with EtOAc.The layers were separated and to the aqueous phase was addedtetrabutylammonium hydrogen sulfate (89 mg, 0.261 mmol). The mixture wasstirred for 30 minutes, then extracted with DCM, dried over sodiumsulfate, and concentrated in vacuo. The crude residue was purified viasilica gel chromatography (MeOH-DCM, 0-30%) affording the title compound(57 mg, 58%) as a colorless film. LC/MS: R_(t)=0.12 min; m/z=322.0 (M+1)Method 2m_acidic.

Step 5: Sodium(4R,5aS,6R,8aS)-6-(methoxymethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylhydrogen sulfate. DOWEX 50Wx8 hydrogen form 200-400 mesh was stirredwith NaOH (2 N) for 3 h then loaded onto a column and washed with wateruntil the pH of the eluant was ˜6, followed by washing withwater-acetone (1:1). Tetrabutylammonium(4R,5aS,6R,8aS)-6-(methoxymethyl)-2,8-dioxohexahydro-2H-1,4-methanopyrrolo[3,4-d][1,3]diazepin-3(4H)-ylsulfate (57 mg, 0.101 mmol) was dissolved in acetone-water (1:1) andpassed through the column, eluting with 1:1 acetone/water. The fractionswere concentrated in vacuo and lyophilized to afford the desired product(27 mg, 70%) as a colorless powder. LC/MS: R_(t)=0.41 min; m/z=321.9(M+1) Method T3_3m_polar; ¹H NMR (500 MHz, D₂O) δ=4.30 (d, J=8.0 Hz,1H), 4.24 (br s, 1H), 3.60-3.56 (m, 1H), 3.56-3.45 (m, 3H), 3.39 (s,3H), 3.38-3.34 (m, 1H), 2.97 (d, J=12.3 Hz, 1H), 2.67 (q, J=8.4 Hz, 1H),2.64-2.56 (m, 1H), 1.72 (dd, J=14.7, 8.4 Hz, 1H).

Susceptibility Testing

MICs were determined by the broth microdilution method in accordancewith Clinical and Laboratories Institute (CLSI) guidelines. In brief,fresh overnight bacterial cultures were suspended in sterile saline, andadjusted to a 0.5 McFarland turbidity standard. Bacterial suspensionswere then diluted in cation adjusted Mueller-Hinton Broth (MHB II; BBL)to yield a final inoculum of approximately 5×10⁵ colony-forming units(CFU)/mL. A master plate of antibiotics was prepared at a concentrationequivalent to hundred-fold the highest desired final concentration in100% dimethyl sulfoxide (DMSO). The master antibiotic plate was thendiluted by serial twofold dilution with a multichannel pipette. Theresulting dilution series of compounds were diluted 1:10 with sterilewater or a solution of beta-lactamase inhibitor prepared at aconcentration equivalent to eleven-fold the desired final concentrationin deionized water leading to a 10% DMSO final concentration. A volumeof 10 μL of the drug dilution series was transferred to 96-well assayplates. Assay plates were inoculated with 90 μL of bacterial suspensionsand incubated at 35° C. for 20 hrs. The assay plates were read using amicrotiter plate reader (Molecular Devices) at 600 nm as well as byvisual observation with a reading mirror. The lowest concentration ofthe compound that prevented visible growth was recorded as the MIC.Performance of the assay was monitored by testing aztreonam againstlaboratory quality control strains in accordance with guidelines of theCLSI.

The following beta-lactamase inhibitors and beta-lactam antibiotics arementioned in the following tables:

Beta-Lactamase Inhibitor 1: Avibactam

Beta-Lactamase Inhibitor 2: Relebactam

Beta-Lactamase Inhibitor 3: Tazobactam

Beta-Lactam 1: Aztreonam

Beta-Lactam 2: Ceftazidime

Beta-Lactam 3: Meropenem

Beta-Lactam 4: Piperacillin

Beta-Lactam 5 (LYS228):

Synergy with Beta-lactams Through Inhibition of Beta-lactamases

Synergy or potentiation of beta-lactam antibiotics through inhibition ofL-lactamases was assessed against an isogenic panel of E. coli strains,each expressing a unique beta-lactamase, and against clinical strains.

Construction of E. coli isogenic strains NB27273-CDY0026 (parent),NB27273-CDY0033 (KPC-2), NB27273-CDY0030 (SHV-12), NB27273-CDY0034(CTX-M-15) and NB27273-CDY0036 (AmpC).

Strain NB27273 (BW25113 pspB::Km^(r)) was obtained from the Keiotransposon insertion collection. The strain has the pspB gene replacedby a kanamycin resistance marker (BW25113 pspB::Km^(r)). This strain wascured of the transposon in pspB via FLP recombinase using publishedmethodology. The resulting strain, BW25113 pspB, was used as a host formulticopy vectors expressing key beta-lactamases. Multicopy plasmidsdirecting constitutive expression of beta-lactamases were established asfollows: Synthetic, codon optimized genes encoding E. coli KPC-2, SHV-12and CTX-M-15 beta-lactamases were made by DNA2.0 (Palo Alto, Calif.).Each of the synthetic fragments were designed to contain NotI and NcoIrestriction sites at their termini, allowing ligation into a NotI/NcoIdigested pET28a(+) derivative for protein expression. The inserts inthese vectors served as template DNA for PCR amplification of the genesencoding KPC-2, SHV-12 and CTX-M-15 using primer pairs E225(tcgcCTCGAGgcgactgcgctgacgaatttgg) (SEQ ID NO:1) and E202(aatcGAATTCttactgaccattaacgcccaagc) (SEQ ID NO:2) and E227(tcgcCTCGAGgcgagcccgcaaccgctgga) (SEQ ID NO:3) and E204(aatcGAATTCttaacgctgccagtgctcaatc) (SEQ ID NO:4) and E226(cgctCTCGAGagcgtcccgctgtacgcacaaacg) (SEQ ID NO:5) and E203,(aatcGAATTCttacagaccgtcggtgacaatc) (SEQ ID NO:6), respectively. Thecodon optimized nucleotide sequences and relevant primer recognitioninformation is shown below:

KPC-2 (SEQ ID NO: 7)ATGGGCCATCATCATCATCATCACAGCAGCGGCCTGGAAGTTCTGTTCCAGGGGCCCGCGACTGCGCT GACGAATTTGGTGGCCGAGCCGTTCGCGAAATTGGAGCAAGATTTTGGTGGTTCGATCGGTGTCTACGCG ATGGACACCGGTAGCGGTGCCACCGTGAGCTACCGTGCCGAAGAGCGTTTTCCGCTGTGTAGCTCTTTCA AGGGTTTTCTGGCCGCAGCCGTGCTGGCACGCAGCCAACAGCAAGCGGGCCTGCTGGACACCCCGATCCG TTACGGCAAAAATGCGCTGGTTCCGTGGAGCCCGATTAGCGAAAAGTACCTGACCACCGGCATGACGGTG GCGGAGTTGAGCGCTGCGGCGGTTCAGTATTCCGATAACGCTGCGGCAAATCTGCTGCTGAAAGAACTGG GCGGTCCAGCGGGTCTGACGGCTTTCATGCGTTCTATTGGCGACACCACCTTTCGCTTGGACCGCTGGGA GCTGGAGCTGAACAGCGCGATTCCGGGCGACGCACGTGATACGAGCAGCCCGCGTGCAGTGACCGAGAGC CTGCAGAAGCTGACCCTGGGCAGCGCACTGGCCGCACCGCAGCGCCAACAGTTCGTCGATTGGCTGAAGG GTAACACCACCGGTAACCATCGTATTCGCGCAGCGGTCCCGGCTGATTGGGCAGTTGGTGACAAGACTGG TACGTGCGGCGTTTATGGTACGGCGAATGACTACGCGGTTGTTTGGCCTACGGGTCGTGCGCCGATCGTC CTGGCGGTGTATACCCGTGCTCCGAACAAAGACGATAAACACTCCGAAGCGGTCATCGCCGCAGCAGCGC GTCTGGCCCTGGAAGGCTTGGGCGTTAATGGTCAGTAACGCCGGCG E225 (SEQ ID NO: 8)TCGCCTCGAGGCGACTGCGCTGACGAATTTGG E202 (SEQ ID NO: 9)AATCGAATTCTTACTGACCATTAACGCCCAAGC REV. COMP. E202 (SEQ ID NO: 10)GCTTGGGCGTTAATGGTCAGTAAGAATTCGATT UNDERLINED = DNA ENCODING BL SHV-12(SEQ ID NO: 11)ATGGGCCATCATCATCATCATCACAGCAGCGGCCTGGAAGTTCTGTTCCAGGGGCCCGCGAGCCCGCAACCGCTGGAGCAGATCAAGCAGTCTGAGAGCCAGCTGAGCGGCCGTGTGGGTATGATCGAGATGGATCTGGCTTCCGGCCGTACGCTGACGGCATGGCGTGCCGACGAACGTTTCCCGATGATGTCGACCTTTAAAGTTGTTCTGTGTGGTGCGGTCTTGGCACGTGTAGACGCGGGTGACGAACAACTGGAGCGCAAGATCCATTACCGCCAACAGGACTTGGTCGACTACAGCCCGGTTAGCGAAAAGCACCTGGCGGATGGCATGACCGTGGGTGAATTGTGCGCCGCTGCGATTACCATGAGCGACAATAGCGCGGCTAATCTGCTGTTGGCGACCGTTGGTGGCCCAGCGGGCTTGACCGCATTTCTGCGTCAAATCGGCGATAATGTTACGCGTCTGGATCGCTGGGAAACGGAGCTGAACGAGGCACTGCCGGGTGATGCCCGTGATACCACGACTCCTGCTAGCATGGCAGCGACCCTGCGTAAACTGCTGACCAGCCAGCGTCTGAGCGCACGTAGCCAACGCCAGCTGCTGCAATGGATGGTGGATGACCGCGTGGCGGGTCCGCTGATCCGCTCCGTCCTGCCAGCAGGCTGGTTCATTGCGGACAAAACTGGTGCCTCTAAGCGTGGTGCGCGTGGTATCGTCGCGCTGCTGGGTCCGAACAACAAAGCCGAACGTATTGTGGTTATCTATCTGCGCGACACCCCGGCAAGCATGGCCGAGCGCAACCAGCAAATTGCGGGCATTGGTGCGGCACTGATTGAGCACTGGCAGCGTTAACGCCGGCG E227(SEQ ID NO: 12) TCGCCTCGAGGCGAGCCCGCAACCGCTGGA E204 (SEQ ID NO: 13)AATCGAATTCTTAACGCTGCCAGTGCTCAATC REV. COMP. E204 (SEQ ID NO: 14)GATTGAGCACTGGCAGCGTTAAGAATTCGATT CTX-M-15ATGGGCCATCATCATCATCATCACAGCAGCGGCCTGGAAGTTCTGTTCCAGGGGCCC AGCGTCCCGCTGTACGCACAAACGGCCGACGTGCAACAGAAACTGGCGGAGTTGGAACGTCAGAGCGGTGGCCGTTTGGGTGTAGCCCTGATCAATACCGCGGACAATAGCCAAATTCTGTATCGTGCGGACGAACGCTTCGCGATGTGCAGCACGAGCAAGGTGATGGCCGCTGCGGCCGTTCTGAAGAAATCCGAGAGCGAGCCGAACTTGCTGAATCAGCGCGTTGAGATCAAGAAGTCGGATCTGGTGAACTATAACCCTATCGCGGAAAAACATGTCAACGGCACCATGTCCCTGGCAGAGCTGAGCGCGGCTGCGTTGCAGTACTCTGATAACGTCGCAATGAATAAACTGATCGCACACGTCGGTGGCCCAGCAAGCGTGACCGCCTTTGCGCGTCAACTGGGCGATGAAACTTTTCGTCTGGATCGTACCGAACCGACCCTGAATACGGCAATTCCGGGTGATCCGCGCGACACGACGAGCCCGCGTGCAATGGCACAGACCCTGCGCAACCTGACCCTGGGTAAAGCGCTGGGCGATAGCCAACGTGCGCAGCTGGTTACGTGGATGAAGGGTAACACCACCGGTGCGGCCAGCATTCAAGCGGGCCTGCCGGCCAGCTGGGTTGTTGGTGATAAAACTGGCTCCGGTGGTTATGGTACCACGAATGACATCGCGGTTATTTGGCCGAAGGACCGTGCGCCGTTGATCCTGGTGACCTACTTCACCCAGCCGCAGCCGAAAGCTGAGTCTCGCCGTGACGTGCTGGCGAGCGCAGCTAAGATTGTCACCGACGGTCTGTAACGCCGGCG E226(SEQ ID NO: 15) cgctCTCGAGagcgtcccgctgtacgcacaaacg E203, (SEQ ID NO: 16)aatcGAATTCttacagaccgtcggtgacaatc

The gene encoding AmpC was PCR amplified from the genome of strain P.aeruginosa PAO1 (NB52019) (GenBank ID U5R279) using primer pair E252(gccCTCGAGggcgaggccccggcggatcgc) (SEQ ID NO: 17) and E253(tgaGAATTCtcagcgcttcagcggcacct) (SEQ ID NO: 18).

The PCR products were then digested with XhoI and EcoRI and ligated intosimilarly digested plasmid pAH63-pstS(BlaP). Plasmid pAH63-pstS(BlaP) isa derivative of plasmid pAH63 (J Bacteriol: 183(21): 6384-6393) made bycloning the TEM-1 (bla) promoter and signal peptide encoding region fromplasmid pBAD (J Bacteriol. 1995 July 177(14):4121-30) into plasmidpAH63. This fragment was PCR amplified from pBAD using primer pair E192(ttcaCTGCAGtgaacgttgcgaagcaacggC) (SEQ ID NO:19) and E194(TCGAggatcctcgagagcaaaaacaggaaggcaaaatgccg) (SEQ ID NO:20), digestedwith PstI and BamHI and inserted into similarly digested plasmid pAH63.Therefore, expression of beta-lactamases from pAH63-pstS(BlaP) basedconstructs is constitutive and the signal sequence is provided to directthese proteins to the periplasm. Plasmid pAH63 based vectors are usedfor insertion into the genome in single copy, however, to provide higherexpression levels to allow more sensitive detection of thesusceptibility of compounds to the expressed beta-lactamases, theexpression inserts contained in these vectors were moved to thereplicative multicopy vector pBAD-Kan (J Bacteriol. 1995 July177(14):4121-30). To accomplish this, the inserts encompassing thebeta-lactamase genes, with the associated TEM promoter and signalsequences, were PCR amplified from their corresponding vectors usingprimer E268 (ccgTCTAGAcggatggcctttttgcgtttc) (SEQ ID NO:21) and E202(aatcGAATTCttactgaccattaacgcccaagc) (SEQ ID NO:22) for the KPC2construct, E204 (aatcGAATTCttaacgctgccagtgctcaatc) (SEQ ID NO:23) forthe SHV-12 construct and E203 (aatcGAATTCttacagaccgtcggtgacaatc) (SEQ IDNO:24) for the CTX-M-15 construct. These fragments were then digestedwith XbaI and EcoRI, and each was inserted into pBAD18-kan that had beendigested with the same enzymes to generate multicopy vectors expressingKPC-2, SHV-12 and CTX-M-15, respectively. These vectors were transformedinto BW25113 pspB to generate strains NB27273-CDY0033 (expressingKPC-2), NB27273-CDY0030 (expressing SHV-12), NB27273-CDY0034 (expressingCTX-M-15) and NB27273-CDY0036 (expressing AmpC). The pBAD18-kan vectoralso contains the TEM promoter region and signal sequence, (but lacksany intact beta-lactamase genes) and was transformed into BW25113 pspBusing standard protocols to generate the control strain NB27273-CDY0026.Expression of the beta-lactamases was confirmed by verifying decreasedsusceptibility to example test antibiotics that are known substrates ofKPC-2, SHV-12, CTX-M-15 or AmpC.

Construction of E. coli isogenic strains, NB27273-CDY0105 (OXA-18) andNB27273-CDY0048 (TEM-10). The plasmid vector for expression of OXA-18,was constructed as follows: The genes encoding GIM-1 (GenBank ID Q704V1)and (OXA-18 (GenBank ID 007293) were synthesized by Life Technologieswith 5′-tgccttcctgtttttgctctcgag and gaattcgctagcccaaaaaaacgg-3′ (SEQ IDNO:25) flanking sequences. The GIM-1 encoding fragment was digested withXhoI and EcoRI and inserted into the KPC-2 expression constructdescribed above from which the KPC-2 encoding gene was removed bydigestion with XhoI and EcoRI. Confirmatory nucleotide sequencingrevealed a XhoI site in the vector backbone which was then removed bysite directed mutagenesis using primer pair E396(cgtcttgctccaggccgcgattaaattcc) (SEQ ID NO:26) and E397(tcgcggcctggagcaagacgtttc) (SEQ ID NO:27). The gene encoding OXA-18 wasthen digested with XhoI and EcoRI and inserted into this vector, fromwhich the gene for GIM-1 had been removed with XhoI and EcoRI.

To generate a vector expressing TEM-10, plasmid pBAD18 (J Bacteriol.1995 July. 177(14):4121-30), which contains the gene encoding TEM-1, wasused as template for PCR based site directed mutagenesis to convert thegene encoding TEM-1 to one encoding TEM-10. From this template DNA,three fragments were generated by PCR using the following primer pairs;

B124 (SEQ ID NO: 28) (tcacgtagcgatagcggag) and E387 (SEQ ID NO: 28)(tggagccggtaagcgtgggtctcgcggt) to generatefragment A encoding an E237K substitution E389 (SEQ ID NO: 29)(cgcgagacccacgcttaccggctccaga) and E391 (SEQ ID NO: 30)(ctcgccttgatagttgggaaccgga) to generatefragment B encoding E237K and R162S substitutions E393 (SEQ ID NO: 31)(cggttcccaactatcaaggcgagt) and E289 SEQ ID NO: 32)(gacattgccgtcactgcgtct) to generatefragment C, also introducing an R162 substitution.

Fragments A, B and C were then used as template to generate a completegene encoding TEM-10 as follows:

Fragments A and B were used as template for PCR using primers B124

(SEQ ID NO: 33) (tcacgtagcgatagcggag) and E390 (SEQ ID NO: 34)(gtaactcgccttgatagttgggaaccggagctgaatgaagc) tocombine fragments A and B into fragment DFragments B and C were used as template for PCR using primers B290

(SEQ ID NO: 35) (gcgggaccaaagccatgaca) and E388 (SEQ ID NO: 36)(accgcgagacccacgcttaccggctccagatttatcagcaataaacc)to combine fragments B and C into fragment E

Finally, Fragments D and E were used as template for PCR using primersE395 (gtaaGAATTCttaccaatgcttaatcagtgaggc) (SEQ ID NO:37) E268(ccgTCTAGAcggatggcctttttgcgtttc) (SEQ ID NO:38) to combine fragments Dand E into the intact TEM-10 encoding product. This fragment was thendigested with XbaI and EcoRI and inserted into pBAD-kan which was alsocut with the same enzymes.

These final vectors for expression of OXA-18 and TEM-10 were transformedinto BW25113 pspB to generate strains NB27273-CDY0105 (expressingOXA-18) and NB27273-CDY0048 (expressing TEM-10). Beta-lactamaseexpression was confirmed by verifying decreased susceptibility toexample test antibiotics that are known substrates of OXA-18 or TEM-10.

TABLE A Minimal Inhibitory Concentrations (MIC), in μg/mL of selectedBLIs E. coli K. pneumoniae P. aeruginosa BLI ATCC 25922 ATCC 43816 ATCC27853 Avibactam 16 32 >64 Relebactam >64 >64 >64 Example 1 >64 >64 >64Example 2 >64 >64 >64 Example 3 >64 >64 >64 Example 4 >64 >64 >64Example 5 >64 >64 >64 Example 6 >64 >64 >64 Example 7 >64 >64 >64Example 8 >64 >64 >64

Table 1 above demonstrates that while some beta-lactamase inhibitorssuch as avibactam exhibit direct antibacterial activity, the compoundsof Formula (A) show little direct activity.

The following data demonstrate the potentiation effect or synergisticactivity of compounds of the invention, as illustrated by the compoundof Example 1, when used in combination with various beta-lactamantibiotics. Since the compound of Example 1 does not exhibit muchdirect antibiotic activity (see Table 1), synergy or potentiation isdefined herein as a four-fold or greater reduction in the MIC of thebeta-lactam antibiotic caused by the presence of the compound of formula(A), compared to the beta-lactam antibiotic alone. Preferably,combinations of the invention exhibit at least an 8-fold reduction inMIC when compared to the beta-lactam antibiotic alone.

Potentiation of Activity (MIC in μg/mL) of Aztreonam by Beta-lactamaseInhibitors in Isogenic Strains of E. coli Expressing IndividualBeta-lactamases.

E. coli E. coli E. coli E. coli E. coli AZTREONAM (KPC- (TEM- (SHV-(CTX-M- E. coli (OXA- (AZ) 2) 10) 12) 15) (AmpC) 18) AZ alone 64 64 >6464 4 >64 AZ + Ex. 1 (2 μg/mL) 0.125 0.125 0.25 ≤0.06 0.125 1 AZ +Avibactam (2 μg/mL) 0.125 0.25 1 0.125 ≤0.06 1 AZ + Relebactam (2 μg/mL)1 2 32 0.5 0.125 >64Potentiation of Activity (M/C in μg/mL) of Ceftazidime by Beta-lactamaseInhibitors in Isogenic Strains of E. coli Expressing IndividualBeta-lactamases.

E. coli E. coli E. coli E. coli CEFTAZIDIME E. coli (TEM- (SHV- (CTX-M-E. coli (OXA- (Ceft) (KPC-2) 10) 12) 15) (AmpC) 18) Ceftazidime Alone4 >64 >64 16 4 >64 Ceft + Ex. 1 (2 μg/mL) 0.125 0.25 0.25 0.25 0.25 0.5Ceft + Avibactam (2 0.25 1 0.5 0.25 0.125 0.5 μg/mL) Ceft + Relebactam(2 0.25 8 8 0.5 0.125 >64 μg/mL)Potentiation of Activity (M/C in μg/mL) of Meropenem by Beta-lactamaseInhibitors in Isogenic Strains of E. coli Expressing IndividualBeta-lactamases.

E. coli E. coli E. coli E. coli E. coli MEROPENEM (KPC- (TEM- (SHV-(CTX-M- E. coli (OXA- (Mero) 2) 10) 12) 15) (AmpC) 18) Mero alone 1≤0.06 ≤0.06 ≤0.06 ≤0.06 ≤0.06 Mero + Ex. 1 (2 μg/mL) ≤0.06 ≤0.06 ≤0.06≤0.06 ≤0.06 ≤0.06 Mero + Avibactam (2 μg/mL) ≤0.06 ≤0.06 ≤0.06 ≤0.06≤0.06 ≤0.06 Mero + Relebactam (2 μg/mL) ≤0.06 ≤0.06 ≤0.06 ≤0.06 ≤0.06≤0.06Potentiation of Activity (MIC in μg/mL) of Piperacillin byBeta-lactamase Inhibitors in Isogenic Strains of E. coli ExpressingIndividual Beta-lactamases.

E. coli E. coli E. coli E. coli E. coli Piperacillin (KPC- (TEM- (SHV-(CTX-M- E. coli (OXA- (Pip) 2) 10) 12) 15) (AmpC) 18) PipAlone >32 >32 >32 >32 >32 >32 Pip + Tazobactam (4 μg/mL) >32 4 32 4 8 8Pip + Avibactam (2 μg/mL) 4 2 4 4 4 2 Pip + Relebactam (2 μg/mL)4 >32 >32 16 4 >32 Pip + Ex. 1 (2 μg/mL) 4 2 2 2 8 2 Pip + Ex. 2 (2μg/mL) 2 4 4 4 8 4 Pip + Ex. 3 (2 μg/mL) 2 4 2 4 8 4 Pip + Ex. 4 (4μg/mL) 2 4 4 2 4 ND Pip + Ex. 5 (4 μg/mL) 1 2 2 2 16 ND Pip + Ex. 6 (4μg/mL) 2 4 4 4 4 ND Pip + Ex. 7 (2 μg/mL) 2 2 4 4 8 8 Pip + Ex. 8 (4μg/mL) 4 4 4 2 4 NDPotentiation of Activity (μg/mL) of Beta-Lactam 5 by Beta-lactamaseInhibitors in Isogenic Strains of E. coli Expressing IndividualBeta-lactamases.

E. coli E. coli E. coli E. coli Beta-Lactam 5 E. coli (TEM- (SHV-(CTX-M- E. coli (OXA- (5) (KPC-2) 10) 12) 15) (AmpC) 18) Beta-Lactam 5Alone 0.25 2 0.5 0.125 0.25 0.5 5 + Ex. 1 (2 μg/mL) 0.25 0.125 0.25 0.250.25 0.25 5 + Avibactam (2 μg/mL) 0.125 0.125 0.125 0.25 0.125 0.125 5 +Relebactam (2 μg/mL) 0.125 0.25 0.125 0.25 0.25 0.25Potentiation of Activity (μg/mL) of Aztreonam by Beta-lactamaseInhibitors in Beta-lactam Resistant Clinical Isolates.

K. pneumoniae Enterobacter cloacae NB29323 NB25044 (CTX-M-15, (CTX-M-12,AZTREONAM OXA-48, VEB-1) ACT, KPC-2) Aztreonam Alone >64 >64 AZ + Ex. 1(2 μg/mL) 0.25 4 AZ + Avibactam (2 μg/mL) 2 8 AZ + Relebactam (2 μg/mL)8 >64Potentiation of Activity (μg/mL) of Piperacillin byBeta-lactamase-inhibitors in Beta-lactam Resistant Clinical Isolates.

Enterobacter K. pneumoniae cloacae S. aureus NB29082 NB25055 NB01437PIPERACILLIN (KPC-2) (CMY-2) (BLA+) Piperacillin >64 >64 64 Pip +Tazobactam (4 μg/mL) >64 64 1 Pip + Ex. 1 (2 μg/mL) 8 4 1

This data demonstrates that potentiation by the compounds of theinvention is similar to or superior to that of some beta-lactamaseinhibitors used in the clinic when it is used in combination withcommercial beta-lactam antibiotics to treat infections caused bybacteria that are resistant to some known beta-lactam antibiotics.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

The invention claimed is:
 1. A compound of Formula (A):

wherein p is 1 or 2; R¹ and R² are H; Z is NR³ ; and R³ is H; or a saltor zwitterionic form thereof.
 2. A compound of claim 1, which is acompound of Formula (I):

wherein: R¹ and R² are H; Z is NR³ ; R³ is H; Y is a cationic group; nis 0 or 1; and when n is 0, the compound of Formula I is in azwitterionic form.
 3. The compound of claim 2, wherein n is 1 and Y isselected from sodium, potassium, ammonium, calcium, magnesium, iron,silver, zinc, and copper.
 4. The compound of claim 3, wherein Y issodium.
 5. The compound of claim 1, which is in a pharmaceuticallyacceptable salt or zwitterionic form.
 6. A compound of Formula (VI):

wherein: R¹ and R² are H; Z is NR³ ; R³ is H; and A is H or —CH₂-Ph,where Ph represents phenyl optionally substituted with one or two groupsselected from halo, C₁-C₄ alkyl, C₁-C₄ alkoxy; or a salt thereof.
 7. Acompound of the formula )VII):

wherein the compound is characterized by XRPD peaks at diffractionangles (2Theta) of 8.3 and 16.6 degrees and one or more additional XRPDpeaks at diffraction angles (2Theta) of 25.1 or 31.3 degrees.
 8. Thecompound of claim 7 in crystalline form.
 9. The compound of claim 8,which exhibits an endotherm on differential scanning calorimetry between283° C. and 350° C.
 10. The compound of claim 7, further characterizedby one or more additional XRPD peaks at diffraction angles (2Theta) of27.4 or 28.7 degrees.
 11. The compound of claim 10, furthercharacterized by additional XRPD peaks at diffraction angles (2Theta) of19.5 degrees or 21.7 degrees.
 12. A process to make a compound ofFormula (I),

according to claim 2; wherein the process comprises contacting acompound of Formula (III)

wherein Z, R¹ and R² and R³ are as defined in claim 2, with asulfonylating agent in the presence of a base.
 13. A pharmaceuticalcomposition comprising the compound of claim 1 and at least onepharmaceutically acceptable excipient.
 14. A method to treat aGram-negative bacterial infection, which comprises administering to asubject in need of such treatment the compound of claims 1 and abeta-lactam antibiotic.
 15. The method of claim 14, wherein thebacterial infection is caused by a species of Burkholderia, Citrobacter,Enterobacter, Escherichia, Klebsiella, Morganella, Moraxella,Providencia, Clostridium, Pseudomonas, Proteus, Salmonella, Serratia,Acinetobacter, Bacteroides, Prevotella, Campylobacter, Neisseria,Enterococcus, Staphylococcus, Streptococcus, Haemophilius orStenotrophomonas bacteria.
 16. The method of claim 14, wherein thebacterial infection is nosocomial pneumonia, an intraabdominalinfection, or a urinary tract infection caused by a species ofEnterobacteriaceae or Pseudomonas.
 17. A method for treating a bacterialinfection comprising administering to a subject in need of suchtreatment the compound of claim
 1. 18. The method of claim 17, whereinthe bacterial infection is caused by a species of Burkholderia,Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella,Moraxella, Providencia, Clostridium, Pseudomonas, Proteus, Salmonella,Serratia, Acinetobacter, Bacteroides, Prevotella, Campylobacter,Neisseria, Enterococcus, Staphylococcus, Streptococcus, Haemophilius orStenotrophomonas.
 19. A pharmaceutical combination, comprising acompound according to claim 1 and a beta-lactam antibiotic.
 20. Themethod of claim 14, wherein the compound is administered in an amounteffective to potentiate the antibacterial activity of the beta-lactamantibiotic.